Secreted and transmembrane polypeptides and nucleic acids encoding the same

ABSTRACT

The present invention is directed to novel polypeptides and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention.

RELATED APPLICATIONS

This application is a continuation of, and claims priority under 35 USC§120 to, U.S. application Ser. No. 09/665350 filed Sep. 18, 2000, whichis a continuation of, and claims priority under 35 USC §120 to, PCTApplication PCT/US00/04414 filed Feb. 22, 2000, which is acontinuation-in-part of, and claims priority under 35 USC §120 to, PCTApplication PCT/US98/19330 filed Sep. 16, 1998, which claims priorityunder 35 USC §119 to U.S. Provisional Application No. 60/063127 filedOct. 24, 1997.

FIELD OF THE INVENTION

The present invention relates generally to the identification andisolation of novel DNA and to the recombinant production of novelpolypeptides.

BACKGROUND OF THE INVENTION

Extracellular proteins play important roles in, among other things, theformation, differentiation and maintenance of multicellular organisms.The fate of many individual cells, e.g., proliferation, migration,differentiation, or interaction with other cells, is typically governedby information received from other cells and/or the immediateenvironment. This information is often transmitted by secretedpolypeptides (for instance, mitogenic factors, survival factors,cytotoxic factors, differentiation factors, neuropeptides, and hormones)which are, in turn, received and interpreted by diverse cell receptorsor membrane-bound proteins. These secreted polypeptides or signalingmolecules normally pass through the cellular secretory pathway to reachtheir site of action in the extracellular environment.

Secreted proteins have various industrial applications, including aspharmaceuticals, diagnostics, biosensors and bioreactors. Most proteindrugs available at present, such as thrombolytic agents, interferons,interleukins, erythropoietins, colony stimulating factors, and variousother cytokines, are secretory proteins. Their receptors, which aremembrane proteins, also have potential as therapeutic or diagnosticagents. Efforts are being undertaken by both industry and academia toidentify new, native secreted proteins. Many efforts are focused on thescreening of mammalian recombinant DNA libraries to identify the codingsequences for novel secreted proteins. Examples of screening methods andtechniques are described in the literature [see, for example, Klein etal., Proc. Natl. Acad. Sci. 93:7108-7113 (1996); U.S. Pat. No.5,536,637)].

Membrane-bound proteins and receptors can play important roles in, amongother things, the formation, differentiation and maintenance ofmulticellular organisms. The fate of many individual cells, e.g.,proliferation, migration, differentiation, or interaction with othercells, is typically governed by information received from other cellsand/or the immediate environment. This information is often transmittedby secreted polypeptides (for instance, mitogenic factors, survivalfactors, cytotoxic factors, differentiation factors, neuropeptides, andhormones) which are, in turn, received and interpreted by diverse cellreceptors or membrane-bound proteins. Such membrane-bound proteins andcell receptors include, but are not limited to, cytokine receptors,receptor kinases, receptor phosphatases, receptors involved in cell-cellinteractions, and cellular adhesin molecules like selectins andintegrins. For instance, transduction of signals that regulate cellgrowth and differentiation is regulated in part by phosphorylation ofvarious cellular proteins. Protein tyrosine kinases, enzymes thatcatalyze that process, can also act as growth factor receptors. Examplesinclude fibroblast growth factor receptor and nerve growth factorreceptor.

Membrane-bound proteins and receptor molecules have various industrialapplications, including as pharmaceutical and diagnostic agents.Receptor immunoadhesins, for instance, can be employed as therapeuticagents to block receptor-ligand interactions. The membrane-boundproteins can also be employed for screening of potential peptide orsmall molecule inhibitors of the relevant receptor/ligand interaction.

Efforts are being undertaken by both industry and academia to identifynew, native receptor or membrane-bound proteins. Many efforts arefocused on the screening of mammalian recombinant DNA libraries toidentify the coding sequences for novel receptor or membrane-boundproteins.

1. PRO211 and PRO217

Epidermal growth factor (EGF) is a conventional mitogenic factor thatstimulates the proliferation of various types of cells includingepithelial cells and fibroblasts. EGF binds to and activates the EGFreceptor (EGFR), which initiates intracellular signaling and subsequenteffects. The EGFR is expressed in neurons of the cerebral cortex,cerebellum, and hippocampus in addition to other regions of the centralnervous system (CNS). In addition, EGF is also expressed in variousregions of the CNS. Therefore, EGF acts not only on mitotic cells, butalso on postmitotic neurons. In fact, many studies have indicated thatEGF has neurotrophic or neuromodulatory effects on various types ofneurons in the CNS. For example, EGF acts directly on cultured cerebralcortical and cerebellar neurons, enhancing neurite outgrowth andsurvival. On the other hand, EGF also acts on other cell types,including septal cholinergic and mesencephalic dopaminergic neurons,indirectly through glial cells. Evidence of the effects of EGF onneurons in the CNS is accumulating, but the mechanisms of action remainessentially unknown. EGF-induced signaling in mitotic cells is betterunderstood than in postmitotic neurons. Studies of clonedpheochromocytoma PC12 cells and cultured cerebral cortical neurons havesuggested that the EGF-induced neurotrophic actions are mediated bysustained activation of the EGFR and mitogen-activated protein kinase(MAPK) in response to EGF. The sustained intracellular signalingcorrelates with the decreased rate of EGFR down-regulation, which mightdetermine the response of neuronal cells to EGF. It is likely that EGFis a multi-potent growth factor that acts upon various types of cellsincluding mitotic cells and postmitotic neurons.

EGF is produced by the salivary and Brunner's glands of thegastrointestinal system, kidney, pancreas, thyroid gland, pituitarygland, and the nervous system, and is found in body fluids such assaliva, blood, cerebrospinal fluid (CSF), urine, amniotic fluid,prostatic fluid, pancreatic juice, and breast milk, Plata-Salaman,Peptides 12: 653-663 (1991).

EGF is mediated by its membrane specific receptor, which contains anintrinsic tyrosine kinase. Stoscheck et al., J. Cell Biochem. 31:135-152 (1986). EGF is believed to function by binding to theextracellular portion of its receptor which induces a transmembranesignal that activates the intrinsic tyrosine kinase.

Purification and sequence analysis of the EGF-like domain has revealedthe presence of six conserved cysteine residues which cross-bind tocreate three peptide loops, Savage et al., J. Biol. Chem. 248: 7669-7672(1979). It is now generally known that several other peptides can reactwith the EGF receptor which share the same generalized motifX_(n)CX₇CX_(4/5)CX₁₀CXCX₅GX₂CX_(n), where X represents any non-cysteineamino acid and n is a variable repeat number. Non isolated peptideshaving this motif include TGF-α, amphiregulin, schwannoma-derived growthfactor (SDGF), heparin-binding EGF-like growth factors and certainvirally encoded peptides (e.g., Vaccinia virus, Reisner, Nature 313:801-803 (1985), Shope fibroma virus, Chang et al., Mol Cell Biol. 7:535-540 (1987), Molluscum contagiosum, Porter and Archard, J. Gen.Virol. 68: 673-682 (1987), and Myxoma virus, Upton et al., J. Virol. 61:1271-1275 (1987), Prigent and Lemoine, Prog. Growth Factor Res. 4: 1-24(1992).

EGF-like domains are not confined to growth factors but have beenobserved in a variety of cell-surface and extracellular proteins whichhave interesting properties in cell adhesion, protein-proteininteraction and development, Laurence and Gusterson, Tumor Biol. 11:229-261 (1990). These proteins include blood coagulation factors(factors VI, IX, X, XII, protein C, protein S, protein Z, tissueplasminogen activator, urokinase), extracellular matrix components(laminin, cytotactin, entactin), cell surface receptors (LDL receptor,thrombomodulin receptor) and immunity-related proteins (complement C1r,uromodulin).

Even more interesting, the general structure pattern of EGF-likeprecursors is preserved through lower organisms as well as in mammaliancells. A number of genes with developmental significance have beenidentified in invertebrates with EGF-like repeats. For example, thenotch gene of Drosophila encodes 36 tandemly arranged 40 amino acidrepeats which show homology to EGF, Wharton et al., Cell 43: 557-581(1985). Hydropathy plots indicate a putative membrane spanning domain,with the EGF-related sequences being located on the extracellular sideof the membrane. Other homeotic genes with EGF-like repeats includeDelta, 95F and 5ZD which were identified using probes based on Notch,and the nematode gene Lin-12 which encodes a putative receptor for adevelopmental signal transmitted between two specified cells.

Specifically, EGF has been shown to have potential in the preservationand maintenance of gastrointestinal mucosa and the repair of acute andchronic mucosal lesions, Konturek et al., Eur. J. Gastroenterol Hepatol.7 (10), 933-37 (1995), including the treatment of necrotizingenterocolitis, Zollinger-Ellison syndrome, gastrointestinal ulcerationgastrointestinal ulcerations and congenital microvillus atrophy,Guglietta and Sullivan, Eur. J. Gastroenterol Hepatol, 7(10), 945-50(1995). Additionally, EGF has been implicated in hair follicledifferentiation; du Cros, J. Invest. Dermatol. 101 (1 Suppl.), 106S-113S(1993), Hillier, Clin. Endocrinol. 33(4), 427-28 (1990); kidneyfunction, Hamm et al., Semin. Nephrol. 13 (1): 109-15 (1993), Harris,Am. J. Kidney Dis. 17(6): 627-30 (1991); tear fluid, van Setten et al.,Int. Ophthalmol 15(6); 359-62 (1991); vitamin K mediated bloodcoagulation, Stenflo et al., Blood 78(7): 1637-51 (1991). EGF is alsoimplicated various skin disease characterized by abnormal keratinocytedifferentiation, e.g., psoriasis, epithelial cancers such as squamouscell carcinomas of the lung, epidermoid carcinoma of the vulva andgliomas. King et al., Am. J. Med. Sci. 296: 154-158 (1988).

Of great interest is mounting evidence that genetic alterations ingrowth factors signaling pathways are closely linked to developmentalabnormalities and to chronic diseases including cancer. Aaronson,Science 254: 1146-1153 (1991). For example, c-erb-2 (also known asHER-2), a proto-oncogene with close structural similarity to EGFreceptor protein, is overexpressed in human breast cancer. King et al.,Science 229: 974-976 (1985); Gullick, Hormones and their actions, Cookeet al., eds, Amsterdam, Elsevier, pp 349-360 (1986).

We herein describe the identification and characterization of novelpolypeptides having homology to EGF, wherein those polypeptides areherein designated PRO211 and PRO217.

2. PRO230

Nephritis is a condition characterized by inflammation of the kidneyaffecting the structure and normal function of the kidney. Thiscondition can be chronic or acute and is generally caused by infection,degenerative process or vascular disease. In all cases, early detectionis desirable so that the patient with nephritis can begin treatment ofthe condition.

An approach to detecting nephritis is to determine the antigensassociated with nephritis and antibodies thereto. In rabbit, atubulointerstitial nephritis antigen (TIN-ag) has been reported inNelson, T. R., et al., J. Biol. Chem., 270(27):16265-70 (July 1995)(GENBANK/U24270). This study reports that the rabbit TIN-ag is abasement membrane glycoprotein having a predicted amino acid sequencewhich has a carboxyl-terminal region exhibiting 30% homology with humanpreprocathepsin B, a member of the cystein proteinase family ofproteins. It is also reported that the rabbit TIN-ag has a domain in theamino-terminal region containing an epidermal growth factor-like motifthat shares homology with laminin A and S chains, alpha 1 chain of typeI collagen, von Willebrand's factor and mucin, indicating structural andfunctional similarities. Studies have also been conducted in mice.However, it is desirable to identify tubulointerstitial nephritisantigens in humans to aid in the development of early detection methodsand treatment of nephritis.

Proteins which have homology to tubulointerstitial nephritis antigensare of particular interest to the medical and industrial communities.Often, proteins having homology to each other have similar function. Itis also of interest when proteins having homology do not have similarfunctions, indicating that certain structural motifs identifyinformation other than function, such as locality of function. We hereindescribe the identification and characterization of a novel polypeptide,designated hgerein as PRO230, which has homology to tubulointerstitialnephritis antigens.

3. PRO232

Stem cells are undifferentiated cells capable of (a) proliferation, (b)self maintenance, (c) the production of a large number of differentiatedfunctional progeny, (d) regeneration of tissue after injury and/or (e) aflexibility in the use of these options. Stem cells often express cellsurface antigens which are capable of serving as cell specific markersthat can be exploited to identify stem cells, thereby providing a meansfor identifying and isolating specific stem cell populations.

Having possession of different stem cell populations will allow for anumber of important applications. For example, possessing a specificstem cell population will allow for the identification of growth factorsand other proteins which are involved in their proliferation anddifferentiation. In addition, there may be as yet undiscovered proteinswhich are associated with (1) the early steps of dedication of the stemcell to a particular lineage, (2) prevention of such dedication, and (3)negative control of stem cell proliferation, all of which may beidentified if one has possession of the stem cell population. Moreover,stem cells are important and ideal targets for gene therapy where theinserted genes promote the health of the individual into whom the stemcells are transplanted. Finally, stem cells may play important roles intransplantation of organs or tissues, for example liver regeneration andskin grafting.

Given the importance of stem cells in various different applications,efforts are currently being undertaken by both industry and academia toidentify new, native stem cell antigen proteins so as to providespecific cell surface markers for identifying stem cell populations aswell as for providing insight into the functional roles played by stemcell antigens in cell proliferation and differentiation. We hereindescribe the identification and characterization of novel polypeptideshaving homology to a stem cell antigen, wherein those polypeptides areherein designated as PRO232 polypeptides.

4. PRO187

Growth factors are molecular signals or mediators that enhance cellgrowth or proliferation, alone or in concert, by binding to specificcell surface receptors. However, there are other cellular reactions thanonly growth upon expression to growth factors. As a result, growthfactors are better characterized as multifunctional and potent cellularregulators. Their biological effects include proliferation, chemotaxisand stimulation of extracellular matrix production. Growth factors canhave both stimulatory and inhibitory effects. For example, transforminggrowth factor (TGF-β) is highly pleiotropic and can stimulateproliferation in some cells, especially connective tissue, while being apotent inhibitor of proliferation in others, such as lymphocytes andepithelial cells.

The physiological effect of growth stimulation or inhibition by growthfactors depends upon the state of development and differentiation of thetarget tissue. The mechanism of local cellular regulation by classicalendocrine molecules involves comprehends autocrine (same cell),juxtacrine (neighbor cell), and paracrine (adjacent cells) pathways.Peptide growth factors are elements of a complex biological language,providing the basis for intercellular communication. They permit cellsto convey information between each other, mediate interaction betweencells and change gene expression. The effect of these multifunctionaland pluripotent factors is dependent on the presence or absence of otherpeptides.

FGF-8 is a member of the fibroblast growth factors (FGFs) which are afamily of heparin-binding, potent mitogens for both normal diploidfibroblasts and established cell lines, Gospodarowicz et al. (1984),Proc. Natl. Acad. Sci. USA 81:6963. The FGF family comprises acidic FGF(FGF-1), basic FGF (FGF-2), INT-2 (FGF-3), K-FGF/HST (FGF-4), FGF-5,FGF-6, KGF (FGF-7), AIGF (FGF-8) among others. All FGFs have twoconserved cysteine residues and share 30-50% sequence homology at theamino acid level. These factors are mitogenic for a wide variety ofnormal diploid mesoderm-derived and neural crest-derived cells,including granulosa cells, adrenal cortical cells, chondrocytes,myoblasts, corneal and vascular endothelial cells (bovine or human),vascular smooth muscle cells, lens, retina and prostatic epithelialcells, oligodendrocytes, astrocytes, chrondocytes, myoblasts andosteoblasts.

Fibroblast growth factors can also stimulate a large number of celltypes in a non-mitogenic manner. These activities include promotion ofcell migration into wound area (chemotaxis), initiation of new bloodvessel formulation (angiogenesis), modulation of nerve regeneration andsurvival (neurotrophism), modulation of endocrine functions, andstimulation or suppression of specific cellular protein expression,extracellular matrix production and cell survival. Baird & Bohlen,Handbook of Exp. Pharmacol. 95(1): 369-418, Springer, (1990). Theseproperties provide a basis for using fibroblast growth factors intherapeutic approaches to accelerate wound healing, nerve repair,collateral blood vessel formation, and the like. For example, fibroblastgrowth factors have been suggested to minimize myocardium damage inheart disease and surgery (U.S. Pat. No. 4,378,347).

FGF-8, also known as androgen-induced growth factor (AIGF), is a 215amino acid protein which shares 30-40% sequence homology with the othermembers of the FGF family. FGF-8 has been proposed to be underandrogenic regulation and induction in the mouse mammary carcinoma cellline SC3. Tanaka et al., Proc. Natl. Acad. Sci. USA 89: 8928-8932(1992); Sato et al., J. Steroid Biochem. Molec. Biol. 47: 91-98 (1993).As a result, FGF-8 may have a local role in the prostate, which is knownto be an androgen-responsive organ. FGF-8 can also be oncogenic, as itdisplays transforming activity when transfected into NIH-3T3fibroblasts. Kouhara et al., Oncogene 9 455-462 (1994). While FGF-8 hasbeen detected in heart, brain, lung, kidney, testis, prostate and ovary,expression was also detected in the absence of exogenous androgens.Schmitt et al., J. Steroid Biochem. Mol. Biol. 57 (34): 173-78 (1996).

FGF-8 shares the property with several other FGFs of being expressed ata variety of stages of murine embryogenesis, which supports the theorythat the various FGFs have multiple and perhaps coordinated roles indifferentiation and embryogenesis. Moreover, FGF-8 has also beenidentified as a protooncogene that cooperates with Wnt-1 in the processof mammary tumorigenesis (Shackleford et al., Proc. Natl. Acad. Sci. USA90, 740-744 (1993); Heikinheimo et al., Mech. Dev. 48: 129-138 (1994)).

In contrast to the other FGFs, FGF-8 exists as three protein isoforms,as a result of alternative splicing of the primary transcript. Tanaka etal., supra. Normal adult expression of FGF-8 is weak and confined togonadal tissue, however northern blot analysis has indicated that FGF-8μmRNA is present from day 10 through day 12 or murine gestation, whichsuggests that FGF-8 is important to normal development. Heikinheimo etal., Mech Dev. 48(2): 129-38 (1994). Further in situ hybridizationassays between day 8 and 16 of indicated initial expression in thesurface ectoderm of the first bronchial arches, the frontonasal process,the forebrain and the midbrain-hindbrain junction. At days 10-12, FGF-8was expressed in the surface ectoderm of the forelimb and hindlimb buds,the nasal its and nasopharynx, the infundibulum and in thetelencephalon, diencephalon and metencephalon. Expression continues inthe developing hindlimbs through day 13 of gestation, but isundetectable thereafter. The results suggest that FGF-8 has a uniquetemporal and spatial pattern in embryogenesis and suggests a role forthis growth factor in multiple regions of ectodermal differentiation inthe post-gastrulation embryo.

We herein describe the identification of novel poypeptides havinghomology to FGF-8, wherein those polypeptides are heein designatedPRO187 polypeptides.

5. PRO265

Protein-protein interactions include receptor and antigen complexes andsignaling mechanisms. As more is known about the structural andfunctional mechanisms underlying protein-protein interactions,protein-protein interactions can be more easily manipulated to regulatethe particular result of the protein-protein interaction. Thus, theunderlying mechanisms of protein-protein interactions are of interest tothe scientific and medical community.

All proteins containing leucine-rich repeats are thought to be involvedin protein-protein interactions. Leucine-rich repeats are short sequencemotifs present in a number of proteins with diverse functions andcellular locations. The crystal structure of ribonuclease inhibitorprotein has revealed that leucine-rich repeats correspond to beta-alphastructural units. These units are arranged so that they form a parallelbeta-sheet with one surface exposed to solvent, so that the proteinacquires an unusual, nonglubular shape. These two features have beenindicated as responsible for the protein-binding functions of proteinscontaining leucine-rich repeats. See, Kobe and Deisenhofer, TrendsBiochem. Sci., 19(10):415-421 (October 1994).

A study has been reported on leucine-rich proteoglycans which serve astissue organizers, orienting and ordering collagen fibrils duringontogeny and are involved in pathological processes such as woundhealing, tissue repair, and tumor stroma formation. Iozzo, R. V., Crit.Rev. Biochem. Mol. Biol., 32(2):141-174 (1997). Others studiesimplicating leucine rich proteins in wound healing and tissue repair areDe La Salle, C., et al., Vouv. Rev. Fr. Hematol. (Germany),37(4):215-222 (1995), reporting mutations in the leucine rich motif in acomplex associated with the bleeding disorder Bernard-Soulier syndromeand Chlemetson, K. J., Thromb. Haemost. (Germany), 74(1): 111-116 (July1995), reporting that platelets have leucine rich repeats. Anotherprotein of particular interest which has been reported to haveleucine-rich repeats is the SLIT protein which has been reported to beuseful in treating neuro-degenerative diseases such as Alzheimer'sdisease, nerve damage such as in Parkinson's disease, and for diagnosisof cancer, see, Artavanistsakonas, S. and Rothberg, J. M., WO9210518-A1by Yale University. Other studies reporting on the biological functionsof proteins having leucine-rich repeats include: Tayar, N., et al., Mol.Cell Endocrinol., (Ireland), 125(1-2):65-70 (December 1996)(gonadotropin receptor involvement); Miura, Y., et al., Nippon Rinsho(Japan), 54(7):1784-1789 (July 1996) (apoptosis involvement); Harris, P.C., et al., J. Am. Soc. Nephrol., 6(4):1125-1133 (October 1995) (kidneydisease involvement); and Ruoslahti, E. I., et al., WO9110727-A by LaJolla Cancer Research Foundation (decorin binding to transforming growthfactor-β involvement for treatment for cancer, wound healing andscarring). Also of particular interest is fibromodulin and its use toprevent or reduce dermal scarring. A study of fibromodulin is found inU.S. Pat. No. 5,654,270 to Ruoslahti, et al.

Efforts are therefore being undertaken by both industry and academia toidentify new proteins having leucine rich repeats to better understandprotein-protein interactions. Of particular interest are those proteinshaving leucine rich repeats and homology to known proteins havingleucine rich repeats such as fibromodulin, the SLIT protein and plateletglycoprotein V. Many efforts are focused on the screening of mammalianrecombinant DNA libraries to identify the coding sequences for novelsecreted and membrane-bound proteins having leucine rich repeats. Weherein describe the identification and characterization of novelpolypeptides having homology to fibromodulin, herein designated asPRO265 polypeptides.

6. PRO219

Human matrilin-2 polypeptide is a member of the von Willebrand factortype A-like module superfamily. von Willebrand factor is a protein whichplays an important role in the maintenence of hemostasis. Morespecifically, von Willebrand factor is a protein which is known toparticipate in platelet-vessel wall interactions at the site of vascularinjury via its ability to interact and form a complex with Factor VIII.The absence of von Willebrand factor in the blood causes an abnormalitywith the blood platelets that prevents platelet adhesion to the vascularwall at the site of the vascular injury. The result is the propensityfor brusing, nose bleeds, intestinal bleeding, and the like comprisingvon Willebrand's disease.

Given the physiological importance of the blood clotting factors,efforts are currently being undertaken by both industry and academia toidentify new, native proteins which may be involved in the coagulationprocess. We herein describe the identification of a novel full-lengthpolypeptide which possesses homology to the human matrilin-2 precursorpolypeptide.

7. PRO246

The cell surface protein HCAR is a membrane-bound protein that acts as areceptor for subgroup C of the adenoviruses and subgroup B of thecoxsackieviruses. Thus, HCAR may provide a means for mediating viralinfection of cells in that the presence of the HCAR receptor on thecellular surface provides a binding site for viral particles, therebyfacilitating viral infection.

In light of the physiological importance of membrane-bound proteins andspecficially those which serve a cell surface receptor for viruses,efforts are currently being undertaken by both industry and academia toidentify new, native membrane-bound receptor proteins. Many of theseefforts are focused on the screening of mammalian recombinant DNAlibraries to identify the coding sequences for novel receptor proteins.We herein describe a novel membrane-bound polypeptide (designated hereinas PRO246) having homology to the cell surface protein HCAR and tovarious tumor antigens including A33 and carcinoembryonic antigen,wherein this polypeptide may be a novel cell surface virus receptor ortumor antigen.

8. PRO228

There are a number of known seven transmembrane proteins and within thisfamily is a group which includes CD97 and EMR1. CD97 is a seven-spantransmembrane receptor which has a cellular ligand, CD55, DAF. Hamann,et al., J. Exp. Med. (U.S.), 184(3):1189 (1996). Additionally, CD97 hasbeen reported as being a dedifferentiation marker in human thyroidcarcinomas and as associated with inflammation. Aust, et al., CancerRes. (U.S.), 57(9):1798 (1997); Gray, et al., J. Immunol. (U.S.),157(12):5438 (1996). CD97 has also been reported as being related to thesecretin receptor superfamily, but unlike known members of that family,CD97 and EMR1 have extended extracellular regions that possess severalEGF domains at the N-terminus. Hamann, et al., Genomics, 32(1):144(1996); Harmann, et al., J. Immunol., 155(4):1942 (1995). EMR1 isfurther described in Lin, et al., Genomics, 41(3):301 (1997) and Baud,et al., Genomics, 26(2):334 (1995). While CD97 and EMR1 appear to berelated to the secretin receptors, a known member of the secretin familyof G protein-coupled receptors includes the alpha-latroxin receptor,latrophilin, which has been described as calcium independent andabundant among neuronal tissues. Lelianova, et al., J. Biol. Chem.,272(34), 21504 (1997); Davletov, et al., J. Biol. Chem. (U.S.),271(38):23239 (1996). Both members of the secretin receptor superfamilyand non-members which are related to the secretin receptor superfamily,or CRF and calcitonin receptors are of interest. In particular, newmembers of these families, identified by their homology to knownproteins, are of interest.

Efforts are being undertaken by both industry and academia to identifynew membrane-bound receptor proteins, particularly transmembraneproteins with EGF repeats and large N-terminuses which may belong to thefamily of seven-transmembrane proteins of which CD97 and EMR1 aremembers. We herein describe the identification and charactization ofnovel polypeptides having homology to CD97 and EMR1, designated hereinas PRO228 polypeptides.

9. PRO533

Growth factors are molecular signals or mediators that enhance cellgrowth or proliferation, alone or in concert, by binding to specificcell surface receptors. However, there are other cellular reactions thanonly growth upon expression to growth factors. As a result, growthfactors are better characterized as multifunctional and potent cellularregulators. Their biological effects include proliferation, chemotaxisand stimulation of extracellular matrix production. Growth factors canhave both stimulatory and inhibitory effects. For example, transforminggrowth factors (TGF-β) is highly pleiotropic and can stimulateproliferation in some cells, especially connective tissues, while beinga potent inhibitor of proliferation in others, such as lymphocytes andepithelial cells.

The physiological effect of growth stimulation or inhibition by growthfactors depends upon the state of development and differentiation of thetarget tissue. The mechanism of local cellular regulation by classicalendocrine molecules comprehends autocrine (same cell), juxtacrine(neighbor cell), and paracrine (adjacent cell) pathways. Peptide growthfactors are elements of a complex biological language, providing thebasis for intercellular communication. They permit cells to conveyinformation between each other, mediate interaction between cells andchange gene expression. The effect of these multifunctional andpluripotent factors is dependent on the presence or absence of otherpeptides.

Fibroblast growth factors (FGFs) are a family of heparin-binding, potentmitogens for both normal diploid fibroblasts and established cell lines,Godpodarowicz, D. et al. (1984), Proc. Natl. Acad. Sci. USA 81: 6983,the FGF family comprises acidic FGF (FGF-1), basic FGF (FGF-2), INT-2(FGF-3), K-FGF/HST (FGF-4), FGF-5, FGF-6, KGF (FGF-7), AIGF (FGF-8)among others. All FGFs have two conserved cysteine residues and share30-50% sequence homology at the amino acid level. These factors aremitogenic for a wide variety of normal diploid mesoderm-derived andneural crest-derived cells, inducing granulosa cells, adrenal corticalcells, chrondocytes, myoblasts, corneal and vascular endothelial cells(bovine or human), vascular smooth muscle cells, lens, retina andprostatic epithelial cells, oligodendrocytes, astrocytes, chrondocytes,myoblasts and osteoblasts.

Fibroblast growth factors can also stimulate a large number of celltypes in a non-mitogenic manner. These activities include promotion ofcell migration into a wound area (chemotaxis), initiation of new bloodvessel formulation (angiogenesis), modulation of nerve regeneration andsurvival (neurotrophism), modulation of endocrine functions, andstimulation or suppression of specific cellular protein expression,extracellular matrix production and cell survival. Baird, A. & Bohlen,P., Handbook of Exp. Phrmacol. 95(1): 369-418 (1990). These propertiesprovide a basis for using fibroblast growth factors in therapeuticapproaches to accelerate wound healing, nerve repair, collateral bloodvessel formation, and the like. For example, fibroblast growth factors,have been suggested to minimize myocardium damage in heart disease andsurgery (U.S. Pat. No. 4,378,437).

We herein describe the identification and characterization of novelpolypeptides having homology to FGF, herein designated PRO533polypeptides.

10. PRO245

Some of the most important proteins involved in the above describedregulation and modulation of cellular processes are the enzymes whichregulate levels of protein phosphorylation in the cell. For example, itis known that the transduction of signals that regulate cell growth anddifferentiation is regulated at least in part by phosphorylation anddephosphorylation of various cellular proteins. The enzymes thatcatalyze these processes include the protein kinases, which function tophosphorylate various cellular proteins, and the protein phosphatases,which function to remove phosphate residues from various cellularproteins. The balance of the level of protein phosphorylation in thecell is thus mediated by the relative activities of these two types ofenzymes.

Although many protein kinase enzymes have been identified, thephysiological role played by many of these catalytic proteins has yet tobe elucidated. It is well known, however, that a number of the knownprotein kinases function to phosphorylate tyrosine residues in proteins,thereby leading to a variety of different effects. Perhaps mostimportantly, there has been a great deal of interest in the proteintyrosine kinases since the discovery that many oncogene products andgrowth factors possess intrinsic protein tyrosine kinase activity. Thereis, therefore, a desire to identify new members of the protein tyrosinekinase family.

Given the physiological importance of the protein kinases, efforts arebeing undertaken by both industry and academia to identify new, nativekinase proteins. Many of these efforts are focused on the screening ofmammalian recombinant DNA libraries to identify the coding sequences fornovel kinase proteins. We herein describe the identification andcharacterization of novel polypeptides having homology to tyrosinekinase proteins, designated herein as PRO245 polypeptides.

11. PRO220, PRO221 and PRO227

Protein-protein interactions include receptor and antigen complexes andsignaling mechanisms. As more is known about the structural andfunctional mechanisms underlying protein-protein interactions,protein-protein interactions can be more easily manipulated to regulatethe particular result of the protein-protein interaction. Thus, theunderlying mechanisms of protein-protein interactions are of interest tothe scientific and medical community.

All proteins containing leucine-rich repeats are thought to be involvedin protein-protein interactions. Leucine-rich repeats are short sequencemotifs present in a number of proteins with diverse functions andcellular locations. The crystal structure of ribonuclease inhibitorprotein has revealed that leucine-rich repeats correspond to beta-alphastructural units. These units are arranged so that they form a parallelbeta-sheet with one surface exposed to solvent, so that the proteinacquires an unusual, nonglubular shape. These two features have beenindicated as responsible for the protein-binding functions of proteinscontaining leucine-rich repeats. See, Kobe and Deisenhofer, TrendsBiochem. Sci., 19(10):415-421 (October 1994).

A study has been reported on leucine-rich proteoglycans which serve astissue organizers, orienting and ordering collagen fibrils duringontogeny and are involved in pathological processes such as woundhealing, tissue repair, and tumor stroma formation. Iozzo, R. V., Crit.Rev. Biochem. Mol. Biol., 32(2):141-174 (1997). Others studiesimplicating leucine rich proteins in wound healing and tissue repair areDe La Salle, C., et al., Vouv. Rev. Fr. Hematol. (Germany),37(4):215-222 (1995), reporting mutations in the leucine rich motif in acomplex associated with the bleeding disorder Bernard-Soulier syndromeand Chlemetson, K. J., Thromb. Haemost. (Germany), 74(1):111-116 (July1995), reporting that platelets have leucine rich repeats. Anotherprotein of particular interest which has been reported to haveleucine-rich repeats is the SLIT protein which has been reported to beuseful in treating neuro-degenerative diseases such as Alzheimer'sdisease, nerve damage such as in Parkinson's disease, and for diagnosisof cancer, see, Artavanistsakonas, S. and Rothberg, J. M., WO9210518-A1by Yale University. Other studies reporting on the biological functionsof proteins having leucine-rich repeats include: Tayar, N., et al., Mol.Cell Endocrinol., (Ireland), 125(1-2):65-70 (December 1996)(gonadotropin receptor involvement); Miura, Y., et al., Nippon Rinsho(Japan), 54(7): 1784-1789 (July 1996) (apoptosis involvement); Harris,P. C., et al., J. Am. Soc. Nephrol., 6(4):1125-1133 (October 1995)(kidney disease involvement); and Ruoslahti, E. I., et al., WO9110727-Aby La Jolla Cancer Research Foundation (decorin binding to transforminggrowth factorβ involvement for treatment for cancer, wound healing andscarring).

Efforts are therefore being undertaken by both industry and academia toidentify new proteins having leucine rich repeats to better understandprotein-protein interactions. Of particular interest are those proteinshaving leucine rich repeats and homology to known proteins havingleucine rich repeats such as the SLIT protein and platelet glycoproteinV.

12. PRO258

Immunoglobulins are antibody molecules, the proteins that function bothas receptors for antigen on the B-cell membrane and as the secretedproducts of the plasma cell. Like all antibody molecules,immunoglobulins perform two major functions: they bind specifically toan antigen and they participate in a limited number of biologicaleffector functions. Therefore, new members of the Ig superfamily arealways of interest. Molecules which act as receptors by various virusesand those which act to regulate immune function are of particularinterest. Also of particular interest are those molecules which havehomology to known Ig family members which act as virus receptors orregulate immune function. Thus, molecules having homology to poliovirusreceptors, CRTAM and CD166 (a ligand for lymphocyte antigen CD6) are ofparticular interest.

Extracellular and membrane-bound proteins play important roles in theformation, differentiation and maintenance of multicellular organisms.The fate of many individual cells, e.g., proliferation, migration,differentiation, or interaction with other cells, is typically governedby information received from other cells and/or the immediateenvironment. This information is often transmitted by secretedpolypeptides (for instance, mitogenic factors, survival factors,cytotoxic factors, differentiation factors, neuropeptides, and hormones)which are, in turn, received and interpreted by diverse cell receptorsor membrane-bound proteins. These secreted polypeptides or signalingmolecules normally pass through the cellular secretory pathway to reachtheir site of action in the extracellular environment, usually at amembrane-bound receptor protein.

We herein describe the identification and characterization of novelpolypeptides having homology to CRTAM, designated herein as PRO258polypeptides.

13. PRO266

Protein-protein interactions include receptor and antigen complexes andsignaling mechanisms. As more is known about the structural andfunctional mechanisms underlying protein-protein interactions,protein-protein interactions can be more easily manipulated to regulatethe particular result of the protein-protein interaction. Thus, theunderlying mechanisms of protein-protein interactions are of interest tothe scientific and medical community.

All proteins containing leucine-rich repeats are thought to be involvedin protein-protein interactions. Leucine-rich repeats are short sequencemotifs present in a number of proteins with diverse functions andcellular locations. The crystal structure of ribonuclease inhibitorprotein has revealed that leucine-rich repeats correspond to beta-alphastructural units. These units are arranged so that they form a parallelbeta-sheet with one surface exposed to solvent, so that the proteinacquires an unusual, nonglobular shape. These two features have beenindicated as responsible for the protein-binding functions of proteinscontaining leucine-rich repeats. See, Kobe and Deisenhofer, TrendsBiochem. Sci., 19(10):415-421 (October 1994).

A study has been reported on leucine-rich proteoglycans which serve astissue organizers, orienting and ordering collagen fibrils duringontogeny and are involved in pathological processes such as woundhealing, tissue repair, and tumor stroma formation. Iozzo, R. V., Crit.Rev. Biochem. Mol. Biol., 32(2):141-174 (1997). Others studiesimplicating leucine rich proteins in wound healing and tissue repair areDe La Salle, C., et al., Vouv. Rev. Fr. Hematol. (Germany),37(4):215-222 (1995), reporting mutations in the leucine rich motif in acomplex associated with the bleeding disorder Bernard-Soulier syndromeand Chlemetson, K. J., Thromb. Haemost. (Germany), 74(1):111-116 (July1995), reporting that platelets have leucine rich repeats. Anotherprotein of particular interest which has been reported to haveleucine-rich repeats is the SLIT protein which has been reported to beuseful in treating neuro-degenerative diseases such as Alzheimer'sdisease, nerve damage such as in Parkinson's disease, and for diagnosisof cancer, see, Artavanistsakonas, S. and Rothberg, J. M., WO9210518-A1by Yale University. Other studies reporting on the biological functionsof proteins having leucine-rich repeats include: Tayar, N., et al., Mol.Cell Endocrinol., (Ireland), 125(1-2):65-70 (December 1996)(gonadotropin receptor involvement); Miura, Y., et al., Nippon Rinsho(Japan), 54(7): 1784-1789 (July 1996) (apoptosis involvement); Harris,P. C., et al., J. Am. Soc. Nephrol., 6(4):1125-1133 (October 1995)(kidney disease involvement); and Ruoslahti, E. I., et al., WO9110727-Aby La Jolla Cancer Research Foundation (decorin binding to transforminggrowth factors involvement for treatment for cancer, wound healing andscarring).

Efforts are therefore being undertaken by both industry and academia toidentify new proteins having leucine rich repeats to better understandprotein-protein interactions, neuronal development and adhesinmolecules. Of particular interest are those proteins having leucine richrepeats and homology to known proteins having leucine rich repeats suchas the SLIT protein. We herein describe novel polypeptides havinghomology to SLIT, designated herein as PRO266 polypeptides.

14. PRO269

Thrombomodulin binds to and regulates the activity of thrombin. It isimportant in the control of blood coagulation. Thrombomodulin functionsas a natural anticoagulant by accelerating the activation of protein Cby thrombin. Soluble thrombomodulin may have therapeutic use as anantithrombotic agent with reduced risk for hemorrhage as compared withheparin. Thrombomodulin is a cell surface trans-membrane glycoprotein,present on endothelial cells and platelets. A smaller, functionallyactive form of thrombomodulin circulates in the plasma and is also foundin urine. (In Haeberli, A., Human Protein Data, VCH Oub., N.Y., 1992).Peptides having homology to thrombomodulin are particularly desirable.

We herein describe the identification and characterization of novelpolypeptides having homology to thrombomodulin, designated herein asPRO269 polypeptides.

15. PRO287

Procollagen C-proteinase enhancer protein binds to and enhances theactivity of bone morphogenic protein “BMP1”/procollagen C-proteinase(PCP). It plays a role in extracellular matrix deposition. BMP1 proteinsmay be used to induce bone and/or cartilage formation and in woundhealing and tissue repair. Therefore, procollagen C-proteinase enhancerprotein, BMP1 and proteins having homology thereto, are of interest tothe scientific and medical communities.

We herein describe the identification and characterization of novelpolypeptides having homology to procollagen C-proteinase enhancerprotein precursor and procollagen C-proteinase enhancer protein,designated herein as PRO287 polypeptides.

16. PRO214

Growth factors are molecular signals or mediators that enhances cellgrowth or proliferation, alone or in concert, by binding to specificcell surface receptors. However, there are other cellular reactions thanonly growth upon expression to growth factors. As a result, growthfactors are better characterized as multifunctional and potent cellularregulators. Their biological effects include proliferation, chemotaxisand stimulation of extracellular matrix production. Growth factors canhave both stimulatory and inhibitory effects. For example, transforminggrowth factor β (TGF-β) is highly pleiotropic and can stimulateproliferation in some cells, especially connective tissue, while being apotent inhibitor of proliferation in others, such as lymphocytes andepithelial cells.

The physiological effect of growth stimulation or inhibition by growthfactors depends upon the state of development and differentiation of thetarget tissue. The mechanism of local cellular regulation by classicalendocrine molecules involves comprehends autocrine (same cell),juxtacrine (neighbor cell), and paracrine (adjacent cells) pathways.Peptide growth factors are elements of a complex biological language,providing the basis for intercellular communication. They permit cellsto convey information between each other, mediate interaction betweencells and change gene expression. The effect of these multifunctionaland pluripotent factors is dependent on the presence or absence of otherpeptides.

Epidermal growth factor (EGF) is a conventional mitogenic factor thatstimulates the proliferation of various types of cells includingepithelial cells and fibroblasts. EGF binds to and activates the EGFreceptor (EGFR), which initiates intracellular signaling and subsequenteffects. The EGFR is expressed in neurons of the cerebral cortex,cerebellum, and hippocampus in addition to other regions of the centralnervous system (CNS). In addition, EGF is also expressed in variousregions of the CNS. Therefore, EGF acts not only on mitotic cells, butalso on postmitotic neurons. In fact, many studies have indicated thatEGF has neurotrophic or neuromodulatory effects on various types ofneurons in the CNS. For example, EGF acts directly on cultured cerebralcortical and cerebellar neurons, enhancing neurite outgrowth andsurvival. On the other hand, EGF also acts on other cell types,including septal cholinergic and mesencephalic dopaminergic neurons,indirectly through glial cells. Evidence of the effects of EGF onneurons in the CNS is accumulating, but the mechanisms of action remainessentially unknown. EGF-induced signaling in mitotic cells is betterunderstood than in postmitotic neurons. Studies of clonedpheochromocytoma PC12 cells and cultured cerebral cortical neurons havesuggested that the EGF-induced neurotrophic actions are mediated bysustained activation of the EGFR and mitogen-activated protein kinase(MAPK) in response to EGF. The sustained intracellular signalingcorrelates with the decreased rate of EGFR down-regulation, which mightdetermine the response of neuronal cells to EGF. It is likely that EGFis a multi-potent growth factor that acts upon various types of cellsincluding mitotic cells and postmitotic neurons.

EGF is produced by the salivary and Brunner's glands of thegastrointestinal system, kidney, pancreas, thyroid gland, pituitarygland, and the nervous system, and is found in body fluids such assaliva, blood, cerebrospinal fluid (CSF), urine, amniotic fluid,prostatic fluid, pancreatic juice, and breast milk, Plata-Salaman, C RPeptides 12: 653-663 (1991).

EGF is mediated by its membrane specific receptor, which contains anintrinsic tyrosine kinase. Stoscheck C M et al., J. Cell Biochem. 31:135-152 (1986). EGF is believed to function by binding to theextracellular portion of its receptor which induces a transmembranesignal that activates the intrinsic tyrosine kinase.

Purification and sequence analysis of the EGF-like domain has revealedthe presence of six conserved cysteine residues which cross-bind tocreate three peptide loops, Savage C R et al., J. Biol. Chem. 248:7669-7672 (1979). It is now generally known that several other peptidescan react with the EGF receptor which share the same generalized motifX_(x)CX₇CX_(4/5)CX₁₀CXCX₅GX₂CX_(n), where X represents any non-cysteineamino acid, and n is a variable repeat number. Non isolated peptideshaving this motif include TGF-a, amphiregulin, schwannoma-derived growthfactor (SDGF), heparin-binding EGF-like growth factors and certainvirally encoded peptides (e.g., Vaccinia virus, Reisner A H, Nature 313:801-803 (1985), Shope fibroma virus, Chang W., et al., Mol Cell Biol. 7:535-540 (1987), Molluscum contagiosum, Porter C D & Archard L C, J. Gen.Virol. 68: 673-682 (1987), and Myxoma virus, Upton C et al., J. Virol.61: 1271-1275 (1987). Prigent S A & Lemoine N. R., Prog. Growth FactorRes. 4: 1-24 (1992).

EGF-like domains are not confined to growth factors but have beenobserved in a variety of cell-surface and extracellular proteins whichhave interesting properties in cell adhesion, protein-proteininteraction and development, Laurence D J R & Gusterson B A, Tumor Biol.11: 229-261 (1990). These proteins include blood coagulation factors(factors VI, IX, X, XII, protein C, protein S, protein Z, tissueplasminogen activator, urokinase), extracellular matrix components(laminin, cytotactin, entactin), cell surface receptors (LDL receptor,thrombomodulin receptor) and immunity-related proteins (complement C1r,uromodulin).

Even more interesting, the general structure pattern of EGF-likeprecursors is preserved through lower organisms as well as in mammaliancells. A number of genes with developmental significance have beenidentified in invertebrates with EGF-like repeats. For example, thenotch gene of Drosophila encodes 36 tandemly arranged 40 amino acidrepeats which show homology to EGF, Wharton W et al., Cell 43: 557-581(1985). Hydropathy plots indicate a putative membrane spanning domain,with the EGF-related sequences being located on the extracellular sideof the membrane. Other homeotic genes with EGF-like repeats includeDelta, 95F and 5ZD which were identified using probes based on Notch,and the nematode gene Lin-12 which encodes a putative receptor for adevelopmental signal transmitted between two specified cells.

Specifically, EGF has been shown to have potential in the preservationand maintenance of gastrointestinal mucosa and the repair of acute andchronic mucosal lesions, Konturek, P C et al., Eur. J. GastroenterolHepatol. 7 (10), 933-37 (1995), including the treatment of necrotizingenterocolitis, Zollinger-Ellison syndrome, gastrointestinal ulcerationgastrointestinal ulcerations and congenital microvillus atrophy, A.Guglietta & P B Sullivan, Eur. J. Gastroenterol Hepatol, 7(10), 945-50(1995). Additionally, EGF has been implicated in hair follicledifferentiation; C. L. du Cros, J. Invest. Dermatol. 101 (1 Suppl.),106S-113S (1993), S G Hillier, Clin. Endocrinol. 33(4),427-28(1990);kidney function, L. L. Hamm et al., Semin. Nephrol. 13 (1): 109-15(1993), R C Harris, Am. J. Kidney Dis. 17(6): 627-30 (1991); tear fluid,G B van Setten et al., Int Ophthalmol 15(6); 359-62 (1991); vitamin Kmediated blood coagulation, J. Stenflo et al., Blood 78(7): 1637-51(1991). EGF is also implicated various skin disease characterized byabnormal keratinocyte differentiation, e.g., psoriasis, epithelialcancers such as squamous cell carcinomas of the lung, epidermoidcarcinoma of the vulva and gliomas. King, L E et al., Am. J. Med. Sci.296: 154-158 (1988).

Of great interest is mounting evidence that genetic alterations ingrowth factors signaling pathways are closely linked to developmentalabnormalities and to chronic diseases including cancer. Aaronson S A,Science 254: 1146-1153 (1991). For example, c-erb-2 (also known asHER-2), a proto-oncogene with close structural similarity to EGFreceptor protein, is overexpressed in human breast cancer. King et al.,Science 229: 974-976 (1985); Gullick, W J, Hormones and their actions,Cooke B A et al., eds, Amsterdam, Elsevier, pp 349-360 (1986).

17. PRO317

The TGF-β supergene family, or simply TGF-β superfamily, a group ofsecreted proteins, includes a large number of related growth anddifferentiation factors expressed in virtually all phyla. Superfamilymembers bind to specific cell surface receptors that activate signaltransduction mechanisms to elicit their multifunctional cytokineeffects. Kolodziejczyk and Hall, Biochem. Cell. Biol., 74: 299-314(1996); Attisano and Wrana, Cytokine Growth Factor Rev., 7: 327-339(1996); and Hill, Cellular Signaling, 8: 533-544 (1996).

Members of this family include five distinct forms of TGF-β (Sporn andRoberts, in Peptide Growth Factors and Their Receptors, Sporn andRoberts, eds. (Springer-Verlag: Berlin, 1990) pp. 419-472), as well asthe differentiation factors vg1 (Weeks and Melton, Cell, 51: 861-867(1987)) and DPP-C polypeptide (Padgett et al., Nature, 325: 81-84(1987)), the hormones activin and inhibin (Mason et al., Nature, 318:659-663 (1985); Mason et al., Growth Factors, 1: 77-88 (1987)), theMullerian-inhibiting substance (MIS) (Cate et al., Cell, 45: 685-698(1986)), the bone morphogenetic proteins (BMPs) (Wozney et al., Science,242: 1528-1534 (1988); PCT WO 88/00205 published Jan. 14, 1988; U.S.Pat. No. 4,877,864 issued Oct. 31, 1989), the developmentally regulatedproteins Vgr-1 (Lyons et al., Proc. Natl. Acad. Sci. USA. 86: 4554-4558(1989)) and Vgr-2 (Jones et al., Molec. Endocrinol., 6: 1961-1968(1992)), the mouse growth differentiation factor (GDF), such as GDF-3and GDF-9 (Kingsley, Genes Dev., 8: 133-146 (1994); McPherron and Lee,J. Biol. Chem., 268: 3444-3449 (1993)), the mouse lefty/Stra1 (Meno etal., Nature, 381: 151-155 (1996); Bouillet et al., Dev. Biol., 170:420-433 (1995)), glial cell line-derived neurotrophic factor (GDNF) (Linet al., Science, 260: 1130-1132 (1993), neurturin (Kotzbauer et al.,Nature, 384: 467-470 (1996)), and endometrial bleeding-associated factor(EBAF) (Kothapalli et al., J. Clin. Invest., 99: 2342-2350 (1997)). Thesubset BMP-2A and BMP-2B is approximately 75% homologous in sequence toDPP-C and may represent the mammalian equivalent of that protein.

The proteins of the TGF-β superfamily are disulfide-linked homo- orheterodimers encoded by larger precursor polypeptide chains containing ahydrophobic signal sequence, a long and relatively poorly conservedN-terminal pro region of several hundred amino acids, a cleavage site(usually polybasic), and a shorter and more highly conserved C-terminalregion. This C-terminal region corresponds to the processed matureprotein and contains approximately 100 amino acids with a characteristiccysteine motif, i.e., the conservation of seven of the nine cysteineresidues of TGF-β among all known family members. Although the positionof the cleavage site between the mature and pro regions varies among thefamily members, the C-terminus of all of the proteins is in theidentical position, ending in the sequence Cys-X-Cys-X, but differing inevery case from the TGF-β consensus C-terminus of Cys-Lys-Cys-Ser. Spornand Roberts, 1990, supra.

There are at least five forms of TGF-β currently identified, TGF-β 1,TGF-β2, TGF-β3, TGF-β4, and TGF-β5. The activated form of TGF-β1 is ahomodimer formed by dimerization of the carboxy-terminal 112 amino acidsof a 390 amino acid precursor. Recombinant TGF-β1 has been cloned(Derynck et al., Nature, 316:701-705 (1985)) and expressed in Chinesehamster ovary cells (Gentry et al., Mol. Cell. Biol., 7: 3418-3427(1987)). Additionally, recombinant human TGF-β2 (deMartin et al., EMBOJ., 6: 3673 (1987)), as well as human and porcine TGF-β3 (Derynck etal., EMBO J., 7: 3737-3743 (1988); ten Dijke et al., Proc. Natl. Acad.Sci. USA, 85: 4715 (1988)) have been cloned. TGF-β2 has a precursor formof 414 amino acids and is also processed to a homodimer from thecarboxy-terminal 112 amino acids that shares approximately 70% homologywith the active form of TGF-β1 (Marquardt et al., J. Biol. Chem., 262:12127 (1987)). See also EP 200,341; 169,016; 268,561; and 267,463; U.S.Pat. No. 4,774,322; Cheifetz et al., Cell, 48: 409-415 (1987); Jakowlewet al., Molecular Endocrin., 2: 747-755 (1988); Derynck et al., J. Biol.Chem., 261: 4377-4379 (1986) Sharples et al., DNA, 6: 239-244 (1987);Derynck et al., Nucl. Acids. Res., 15: 3188-3189 (1987); Derynck et al.,Nucl. Acids. Res., 15: 3187 (1987); Seyedin et al., J. Biol. Chem., 261:5693-5695 (1986); Madisen et al., DNA, 7: 1-8 (1988); and Hanks et al.,Proc. Natl. Acad. Sci. (U.S.A.), 85: 79-82 (1988).

TGF-β4 and TGF-β5 were cloned from a chicken chondrocyte cDNA library(Jakowlew et al., Molec. Endocrinol., 2: 1186-1195 (1988)) and from afrog oocyte cDNA library, respectively.

The pro region of TGF-β associates non-covalently with the mature TGF-βdimer (Wakefield et al., J. Biol. Chem., 263: 7646-7654 (1988);Wakefield et al., Growth Factors, 1: 203-218 (1989)), and the proregions are found to be necessary for proper folding and secretion ofthe active mature dimers of both TGF-β and activin (Gray and Mason,Science, 247: 1328-1330 (1990)). The association between the mature andpro regions of TGF-β masks the biological activity of the mature dimer,resulting in formation of an inactive latent form. Latency is not aconstant of the TGF-β superfamily, since the presence of the pro regionhas no effect on activin or inhibin biological activity.

A unifying feature of the biology of the proteins from the TGF-βsuperfamily is their ability to regulate developmental processes. TGF-βhas been shown to have numerous regulatory actions on a wide variety ofboth normal and neoplastic cells. TGF-β is multifunctional, as it caneither stimulate or inhibit cell proliferation, differentiation, andother critical processes in cell function (Sporn and Roberts, supra).

One member of the TGF-β superfamily, EBAF, is expressed in endometriumonly in the late secretory phase and during abnormal endometrialbleeding. Kothapalli et al., J. Clin. Invest., 99: 2342-2350 (1997).Human endometrium is unique in that it is the only tissue in the bodythat bleeds at regular intervals. In addition, abnormal endometrialbleeding is one of the most common manifestations of gynecologicaldiseases, and is a prime indication for hysterectomy. In situhybridization showed that the mRNA of EBAF was expressed in the stromawithout any significant mRNA expression in the endometrial glands orendothelial cells.

The predicted protein sequence of EBAF showed a strong homology to theprotein encoded by mouse lefty/stra3 of the TGF-β superfamily. A motifsearch revealed that the predicted EBAF protein contains most of thecysteine residues which are conserved among the TGF-β-related proteinsand which are necessary for the formation of the cysteine knotstructure. The EBAF sequence contains an additional cysteine residue, 12amino acids upstream from the first conserved cysteine residue. The onlyother family members known to contain an additional cysteine residue areTGF-βs, inhibins, and GDF-3. EBAF, similar to LEFTY, GDF-3/Vgr2, andGDF-9, lacks the cysteine residue that is known to form theintermolecular disulfide bond. Therefore, EBAF appears to be anadditional member of the TGF-β superfamily with an unpaired cysteineresidue that may not exist as a dimer. However, hydrophobic contactsbetween the two monomer subunits may promote dimer formation.Fluorescence in situ hybridization showed that the ebaf gene is locatedon human chromosome 1 at band q42.1.

Additional members of the TGF-β superfamily, such as those related toEBAF, are being searched for by industry and academics. We hereindescribe the identification and characterization of novel polypeptideshaving homology to EBAF, designated herein as PRO317 polypeptides.

18. PRO301

The widespread occurrence of cancer has prompted the devotion ofconsiderable resources and discovering new treatments of treatment. Oneparticular method involves the creation of tumor or cancer specificmonoclonal antibodies (mAbs) which are specific to tumor antigens. SuchmAbs, which can distinguish between normal and cancerous cells areuseful in the diagnosis, prognosis and treatment of the disease.Particular antigens are known to be associated with neoplastic diseases,such as colorectal cancer.

One particular antigen, the A33 antigen is expressed in more than 90% ofprimary or metastatic colon cancers as well as normal colon epithelium.Since colon cancer is a widespread disease, early diagnosis andtreatment is an important medical goal. Diagnosis and treatment of coloncancer can be implemented using monoclonal antibodies (mAbs) specifictherefore having fluorescent, nuclear magnetic or radioactive tags.Radioactive gene, toxins and/or drug tagged mAbs can be used fortreatment in situ with minimal patient description. mAbs can also beused to diagnose during the diagnosis and treatment of colon cancers.For example, when the serum levels of the A33 antigen are elevated in apatient, a drop of the levels after surgery would indicate the tumorresection was successful. On the other hand, a subsequent rise in serumA33 antigen levels after surgery would indicate that metastases of theoriginal tumor may have formed or that new primary tumors may haveappeared. Such monoclonal antibodies can be used in lieu of, or inconjunction with surgery and/or other chemotherapies. For example, U.S.Pat. No. 4,579,827 and U.S. Ser. No. 424,991 (E.P. 199,141) are directedto therapeutic administration of monoclonal antibodies, the latter ofwhich relates to the application of anti-A33 mAb.

Many cancers of epithelial origin have adenovirus receptors. In fact,adenovirus-derived vectors have been proposed as a means of insertingantisense nucleic acids into tumors (U.S. Pat. No. 5,518,885). Thus, theassociation of viral receptors with neoplastic tumors is not unexpected.

We herein describe the identification and characterization of novelpolypeptides having homology to certain cancer-associated antigens,designated herein as PRO301 polypeptides.

19. PRO224

Cholesterol uptake can have serious implications on one's health.Cholesterol uptake provides cells with most of the cholesterol theyrequire for membrane synthesis. If this uptake is blocked, cholesterolaccumulates in the blood and can contribute to the formation ofatherosclerotic plaques in blood vessel walls. Most cholesterol istransported in the blood bound to protein in the form of complexes knownas low-density lipoproteins (LDLs). LDLs are endocytosed into cells viaLDL receptor proteins. Therefore, LDL receptor proteins, and proteinshaving homology thereto, are of interest to the scientific and medicalcommunities.

Membrane-bound proteins and receptors can play an important role in theformation, differentiation and maintenance of multicellular organisms.The LDL receptors are an example of membrane-bound proteins which areinvolved in the synthesis and formation of cell membranes, wherein thehealth of an individual is affected directly and indirectly by itsfunction. Many membrane-bound proteins act as receptors such as the LDLreceptor. These receptors can function to endocytose substrates or theycan function as a receptor for a channel. Other membrane-bound proteinsfunction as signals or antigens.

Membrane-bound proteins and receptor molecules have various industrialapplications, including as pharmaceutical and diagnostic agents. Themembrane-bound proteins can also be employed for screening of potentialpeptide or small molecule regulators of the relevant receptor/ligandinteraction. In the case of the LDL receptor, it is desirable to findmolecules which enhance endocytosis so as to lower blood cholesterollevels and plaque formation. It is also desirable to identify moleculeswhich inhibit endocytosis so that these molecules can be avoided orregulated by individuals having high blood cholesterol. Polypeptideswhich are homologous to lipoprotein receptors but which do not functionas lipoprotein receptors are also of interest in the determination ofthe function of the fragments which show homology.

The following studies report on previously known low density lipoproteinreceptors and related proteins including apolipoproteins: Sawamura, etal., Nippon Chemiphar Co, Japan patent application J09098787; Novak, S.,et al., J. Biol. Chem., 271:(20)11732-6 (1996); Blaas, D., J. Virol.,69(11)7244-7 (November 1995); Scott, J., J. Inherit. Metab. Dis. (UK),9/Supp. 1 (3-16) (1986); Yamamoto, et al., Cell, 39:27-38 (1984);Rebece, et al., Neurobiol. Aging, 15:5117(1994); Novak, S., et al., J.Biol. Chemistry, 271:11732-11736(1996); and Sestavel and Fruchart, CellMol. Biol., 40(4):461-81 (June 1994). These publications and otherspublished prior to the filing of this application provide furtherbackground to peptides already known in the art.

Efforts are being undertaken by both industry and academia to identifynew, native membrane-bound receptor proteins, particularly those havinghomology to lipoprotein receptors. We herein describe the identificationand characterization of novel polypeptides having homology tolipoprotein receptors, designated herein as PRO224 polypeptides.

20. PRO222

Complement is a group of proteins found in the blood that are importantin humoral immunity and inflammation. Complement proteins aresequentially activated by antigen-antibody complexes or by proteolyticenzymes. When activated, complement proteins kill bacteria and othermicroorganisms, affect vascular permeability, release histamine andattract white blood cells. Complement also enhances phagocytosis whenbound to target cells. In order to prevent harm to autologous cells, thecomplement activation pathway is tightly regulated.

Deficiencies in the regulation of complement activation or in thecomplement proteins themselves may lead to immune-complex diseases, suchas systemic lupus erythematosus, and may result in increasedsusceptibility to bacterial infection. In all cases, early detection ofcomplement deficiency is desirable so that the patient can begintreatment. Thus, research efforts are currently directed towardidentification of soluble and membrane proteins that regulate complementactivation.

Proteins known to be important in regulating complement activation inhumans include Factor H and Complement receptor type 1 (CR1). Factor His a 150 kD soluble serum protein that interacts with complement proteinC3b to accelerate the decay of C3 convertase and acts as a cofactor forFactor I-mediated cleavage of complement protein C4b. Complementreceptor type 1 is a 190-280 kD membrane bound protein found in mastcells and most blood cells. CR1 interacts with complement proteins C3b,C4b, and C3b to accelerate dissociation of C3 convertases, acts as acofactor for Factor I-mediated cleavage of C3b and C4b, and binds immunecomplexes and promotes their dissolution and phagocytosis.

Proteins which have homology to complement proteins are of particularinterest to the medical and industrial communities. Often, proteinshaving homology to each other have similar function. It is also ofinterest when proteins having homology do not have similar functions,indicating that certain structural motifs identify information otherthan function, such as locality of function.

Efforts are being undertaken by both industry and academia to identifynew, native secreted and membrane-bound proteins, particularly thosehaving homology to known proteins involved in the complement pathway.Proteins involved in the complement pathway were reviewed in BirminghamD J (1995), Critical Reviews in Immunology, 15(2):133-154 and in Abbas AK, et al. (1994) Cellular and Molecular Immunology, 2nd Ed. W. B.Saunders Company, Philadelphia, pp 295-315.

We herein describe the identification and characterization of novelpolypeptides having homology to complement receptors, designated hereinas PRO222 polypeptides.

21. PRO234

The successful function of many systems within multicellular organismsis dependent on cell-cell interactions. Such interactions are affectedby the alignment of particular ligands with particular receptors in amanner which allows for ligand-receptor binding and thus a cell-celladhesion. While protein-protein interactions in cell recognition havebeen recognized for some time, only recently has the role ofcarbohydrates in physiologically relevant recognition been widelyconsidered (see B. K. Brandley et al., J. Leuk. Biol. 40: 97 (1986) andN. Sharon et al., Science 246: 227 (1989). Oligosaccharides are wellpositioned to act as recognition novel lectins due to their cell surfacelocation and structural diversity. Many oligosaccharide structures canbe created through the differential activities of a smaller number ofglycosyltransferases. The diverse structures of oligosaccharides can begenerated by transcription of relatively few gene products, whichsuggests that the oligosaccharides are a plausible mechanism by which isdirected a wide range of cell-cell interactions. Examples ofdifferential expression of cell surface carbohydrates and putativecarbohydrate binding proteins (lectins) on interacting cells have beendescribed (J. Dodd & T. M. Jessel, J. Neurosci. 5: 3278 (1985); L. J.Regan et al., Proc. Natl. Acad. Sci. USA 83: 2248 (1986); M.Constantine-Paton et al., Nature 324: 459 (1986); and M. Tiemeyer etal., J. Biol. Chem. 263: 1671 (1989). One interesting member of thelectin family are selecting.

The migration of leukocytes to sites of acute or chronic inflammationinvolves adhesive interactions between these cells and the endothelium.This specific adhesion is the initial event in the cascade that isinitiated by inflammatory insults, and it is, therefore, of paramountimportance to the regulated defense of the organism.

The types of cell adhesion molecules that are involved in theinteraction between leukocytes and the endothelium during aninflammatory response currently stands at four: (1) selectins; (2)(carbohydrate and glycoprotein) ligands for selecting; (3) integrins;and (4) integrin ligands, which are members of the immunoglobulin genesuperfamily.

The selectins are cell adhesion molecules that are unified bothstructurally and functionally. Structurally, selectins are characterizedby the inclusion of a domain with homology to a calcium-dependent lectin(C-lectins), an epidermal growth factor (egf)-like domain and severalcomplement binding-like domains, Bevilacqua, M. P. et al., Science 243:1160-1165 (1989); Johnston et al., Cell 56: 1033-1044 (1989); Lasky etal, Cell 56: 1045-1055 (1989); Siegalman, M. et al., Science 243:1165-1172 (1989); Stoolman, L. M., Cell 56: 907-910 (1989).Functionally, selectins share the common property of their ability tomediate cell binding through interactions between their lectin domainsand cell surface carbohydrate ligands (Brandley, B, et al., Cell 63,861-863 (1990); Springer, T. and Lasky, L. A., Nature 349 19-197 (1991);Bevilacqua, M. P. and Nelson, R. M., J. Clin. Invest. 91 379-387 (1993)and Tedder et al., J. Exp. Med. 170: 123-133 (1989).

There are three members identified so far in the selectin family of celladhesion molecules: L-selectin (also called peripheral lymph node homingreceptor (pnHR), LEC-CAM-1, LAM-1, gp90^(MEL), gp100^(MEL), gp10^(MEL),MEL-14 antigen, Leu-8 antigen, TQ-1 antigen, DREG antigen), E-selectin(LEC-CAM-2, LECAM-2, ELAM-1) and P-selectin (LEC-CAM-3, LECAM-3,GMP-140, PADGEM).

The identification of the C-lectin domain has led to an intense effortto define carbohydrate binding ligands for proteins containing suchdomains. E-selectin is believed to recognize the carbohydrate sequenceNeuNAcα2-3Galβ1-4(Fucα1-3) GlcNAc (sialyl-Lewis x, or sLe^(x)) andrelated oligosaccharides, Berg et al., J. Biol. Chem. 265: 14869-14872(1991); Lowe et al., Cell 63: 475-484 (1990); Phillips et al., Science250: 1130-1132(1990); Tiemeyer et al., Proc. Natl. Acad. Sci. USA 88:1138-1142(1991).

L-selectin, which comprises a lectin domain, performs its adhesivefunction by recognizing carbohydrate-containing ligands on endothelialcells. L-selectin is expressed on the surface of leukocytes, such aslymphocytes, neutrophils, monocytes and eosinophils, and is involvedwith the trafficking of lymphocytes to peripheral lymphoid tissues(Gallatin et al., Nature 303: 30-34 (1983)) and with acuteneutrophil-medicated inflammatory responses (Watson, S. R., Nature 349:164-167 (1991)). The amino acid sequence of L-selectin and the encodingnucleic acid sequence are, for example, disclosed in U.S. Pat. No.5,098,833 issued 24 Mar. 1992.

L-selectin (LECAM-1) is particularly interesting because of its abilityto block neutrophil influx (Watson et al., Nature 349: 164-167 (1991).It is expressed in chronic lymphocytic leukemia cells which bind to HEV(Spertini et al., Nature 349: 691-694 (1991). It is also believed thatHEV structures at sites of chronic inflammation are associated with thesymptoms of diseases such as rheumatoid arthritis, psoriasis andmultiple sclerosis.

E-selectin (ELAM-1), is particularly interesting because of itstransient expression on endothelial cells in response to IL-1 or TNF.Bevilacqua et al., Science 243: 1160 (1989). The time course of thisinduced expression (2-8 h) suggests a role for this receptor in initialneutrophil induced extravasation in response to infection and injury. Ithas further been reported that anti-ELAM-1 antibody blocks the influx ofneutrophils in a primate asthma model and thus is beneficial forpreventing airway obstruction resulting from the inflammatory response.Gundel et al., J. Clin. Invest. 88: 1407 (1991).

The adhesion of circulating neutrophils to stimulated vascularendothelium is a primary event of the inflammatory response. P-selectinhas been reported to recognize the Lewis x structure (Galβ1-4(Fucα1-3)GlcNAc), Larsen et al., Cell 63: 467-474(1990). Others report that anadditional terminal linked sialic acid is required for high affinitybinding, Moore et al., J. Cell. Biol. 112: 491-499 (1991). P-selectinhas been shown to be significant in acute lung injury. Anti-P-selectinantibody has been shown to have strong protective effects in a rodentlung injury model. M. S. Mulligan et al., J. Clin. Invest. 90: 1600(1991).

We herein describe the identification and characterization of novelpolypeptides having homology to lectin proteins, herein designated asPRO234 polypeptides.

22. PRO231

Some of the most important proteins involved in the above describedregulation and modulation of cellular processes are the enzymes whichregulate levels of protein phosphorylation in the cell. For example, itis known that the transduction of signals that regulate cell growth anddifferentiation is regulated at least in part by phosphorylation anddephosphorylation of various cellular proteins. The enzymes thatcatalyze these processes include the protein kinases, which function tophosphorylate various cellular proteins, and the protein phosphatases,which function to remove phosphate residues from various cellularproteins. The balance of the level of protein phosphorylation in thecell is thus mediated by the relative activities of these two types ofenzymes.

Protein phosphatases represent a growing family of enzymes that arefound in many diverse forms, including both membrane-bound and solubleforms. While many protein phosphatases have been described, thefunctions of only a very few are beginning to be understood (Tonks,Semin. Cell Biol. 4:373-453 (1993) and Dixon, Recent Prog. Horm. Res.51:405-414 (1996)). However, in general, it appears that many of theprotein phosphatases function to modulate the positive or negativesignals induced by various protein kinases. Therefore, it is likely thatprotein phosphatases play critical roles in numerous and diversecellular processes.

Given the physiological importance of the protein phosphatases, effortsare being undertaken by both industry and academia to identify new,native phosphatase proteins. Many of these efforts are focused on thescreening of mammalian recombinant DNA libraries to identify the codingsequences for novel phosphatase proteins. Examples of screening methodsand techniques are described in the literature [see, for example, Kleinet al., Proc. Natl. Acad. Sci., 93:7108-7113 (1996); U.S. Pat. No.5,536,637)].

We herein describe the identification and characterization of novelpolypeptides having homology to acid phosphatases, designated herein asPRO231 polypeptides.

23. PRO229

Scavenger receptors are known to protect IgG molecules from catabolicdegradation. Riechmann and Hollinger, Nature Biotechnology, 15:617(1997). In particular, studies of the CH2 and CH3 domains have shownthat specific sequences of these domains are important in determiningthe half-lives of antibodies. Ellerson, et al., J. Immunol., 116: 510(1976); Yasmeen, et al., J. Immunol. 116: 518 (1976; Pollock, et al.,Eur. J. Immunol., 20: 2021 (1990). Scavenger receptor proteins andantibodies thereto are further reported in U.S. Pat. No. 5,510,466 toKrieger, et al. Due to the ability of scavenger receptors to increasethe half-life of polypeptides and their involvement in immune function,molecules having homology to scavenger receptors are of importance tothe scientific and medical community.

Efforts are being undertaken by both industry and academia to identifynew, native secreted and membrane-bound receptor proteins, particularlythose having homology to scavenger receptors. Many efforts are focusedon the screening of mammalian recombinant DNA libraries to identify thecoding sequences for novel secreted and membrane-bound receptorproteins. Examples of screening methods and techniques are described inthe literature [see, for example, Klein et al., Proc. Natl. Acad. Sci.,93:7108-7113 (1996); U.S. Pat. No. 5,536,637)].

We herein describe the identification and characterization of novelpolypeptides having homology to scavenger receptors, designated hereinas PRO229 polypeptides.

24. PRO238

Oxygen free radicals and antioxidants appear to play an important rolein the central nervous system after cerebral ischemia and reperfusion.Moreover, cardiac injury, related to ischaemia and reperfusion has beenreported to be caused by the action of free radicals. Additionally,studies have reported that the redox state of the cell is a pivotaldeterminant of the fate of the cells. Furthermore, reactive oxygenspecies have been reported to be cytotoxic, causing inflammatorydisease, including tissue necrosis, organ failure, atherosclerosis,infertility, birth defects, premature aging, mutations and malignancy.Thus, the control of oxidation and reduction is important for a numberof reasons including for control and prevention of strokes, heartattacks, oxidative stress and hypertension. In this regard, reductases,and particularly, oxidoreductases, are of interest. Publications furtherdescribing this subject matter include Kelsey, et al., Br. J. Cancer,76(7):8524 (1997); Friedrich and Weiss, J. Theor. Biol., 187(4):529-40(1997) and Pieulle, et al., J. Bacteriol., 179(18):5684-92 (1997).

Efforts are being undertaken by both industry and academia to identifynew, native secreted and membrane-bound receptor proteins, particularlysecreted proteins which have homology to reductase. Many efforts arefocused on the screening of mammalian recombinant DNA libraries toidentify the coding sequences for novel secreted and membrane-boundreceptor proteins. Examples of screening methods and techniques aredescribed in the literature [see, for example, Klein et al., Proc. Natl.Acad. Sci., 93:7108-7113 (1996); U.S. Pat. No. 5,536,637)].

We herein describe the identification and characterization of novelpolypeptides having homology to reductase, designated herein as PRO238polypeptides.

25. PRO233

Studies have reported that the redox state of the cell is an importantdeterminant of the fate of the cell. Furthermore, reactive oxygenspecies have been reported to be cytotoxic, causing inflammatorydisease, including tissue necrosis, organ failure, atherosclerosis,infertility, birth defects, premature aging, mutations and malignancy.Thus, the control of oxidation and reduction is important for a numberof reasons, including the control and prevention of strokes, heartattacks, oxidative stress and hypertension. Oxygen free radicals andantioxidants appear to play an important role in the central nervoussystem after cerebral ischemia and reperfusion. Moreover, cardiacinjury, related to ischaemia and reperfusion has been reported to becaused by the action of free radicals. In this regard, reductases, andparticularly, oxidoreductases, are of interest. In addition, thetranscription factors, NF-kappa B and AP-1, are known to be regulated byredox state and to affect the expression of a large variety of genesthought to be involved in the pathogenesis of AIDS, cancer,atherosclerosis and diabetic complications. Publications furtherdescribing this subject matter include Kelsey, et al., Br. J. Cancer,76(7):852-4 (1997); Friedrich and Weiss, J. Theor. Biol., 187(4):529-40(1997) and Pieulle, et al., J. Bacteriol., 179(18):5684-92 (1997). Giventhe physiological importance of redox reactions in vivo, efforts arecurrently being under taken to identify new, native proteins which areinvolved in redox reactions. We describe herein the identification ofnovel polypeptides which have homology to reductase, designated hereinas PRO233 polypeptides.

26. PRO223

The carboxypeptidase family of exopeptidases constitutes a diverse groupof enzymes that hydrolyze carboxyl-terminal amide bonds in polypeptides,wherein a large number of mammalian tissues produce these enzymes. Manyof the carboxypeptidase enzymes that have been identified to dateexhibit rather strong cleavage specificities for certain amino acids inpolypeptides. For example, carboxypeptidase enzymes have been identifiedwhich prefer lysine, arginine, serine or amino acids with eitheraromatic or branched aliphatic side chains as substrates at the carboxylterminus of the polypeptide.

With regard to the serine carboxypeptidases, such amino acid specificenzymes have been identified from a variety of different mammalian andnon-mammalian organisms. The mammalian serine carboxypeptidase enzymesplay important roles in many different biological processes including,for example, protein digestion, activation, inactivation, or modulationof peptide hormone activity, and alteration of the physical propertiesof proteins and enzymes.

In light of the physiological importance of the serinecarboxypeptidases, efforts are being undertaken by both industry andacademia to identify new, native secreted and membrane-bound receptorproteins and specifically novel carboxypeptidases. Many of these effortsare focused on the screening of mammalian recombinant DNA libraries toidentify the coding sequences for novel secreted and membrane-boundreceptor proteins. We describe herein novel polypeptides having homologyto one or more serine carboxypeptidase polypeptides, designated hereinas PRO223 polypeptides.

27. PRO235

Plexin was first identified in Xenopus tadpole nervous system as amembrane glycoprotein which was shown to mediate cell adhesion via ahomophilic binding mechanism in the presence of calcium ions. Strongevolutionary conservation between Xenopus, mouse and human homologs ofplexin has been observed. [Kaneyama et al., Biochem. And Biophys. Res.Comm. 226: 524-529 (1996)]. Given the physiological importance of celladhesion mechanisms in vivo, efforts are currently being under taken toidentify new, native proteins which are involved in cell adhesion. Wedescribe herein the identification of a novel polypeptide which hashomology to plexin, designated herein as PRO235.

28. PRO236 and PRO262

β-galactosidase is a well known enzymatic protein which functions tohydrolyze β-galactoside molecules. β-galactosidase has been employed fora variety of different applications, both in vitro and in vivo and hasproven to be an extremely useful research tool. As such, there is aninterest in obtaining novel polypeptides which exhibit homology to theβ-galactosidase polypeptide.

Given the strong interest in obtaining novel polypeptides havinghomology to β-galactosidase, efforts are currently being undertaken byboth industry and academia to identify new, native β-galactosidasehomolog proteins. Many of these efforts are focused on the screening ofmammalian recombinant DNA libraries to identify the coding sequences fornovel β-galactosidase-like proteins. Examples of screening methods andtechniques are described in the literature [see, for example, Klein etal., Proc. Natl. Acad. Sci., 93:7108-7113 (1996); U.S. Pat. No.5,536,637)]. We herein describe novel poylpeptides having siginificanthomology to the β-galactosidase enzyme, designated herein as PRO236 andPRO262 polypeptides.

29. PRO239

Densin is a glycoprotein which has been isolated from the brain whichhas all the hallmarks of an adhesion molecule. It is highly concentratedat synaptic sites in the brain and is expressed prominently in dendriticprocesses in developing neurons. Densin has been characterized as amember of the O-linked sialoglycoproteins. Densin has relevance tomedically important processes such as regeneration. Given thephysiological importance of synaptic processes and cell adhesionmechanisms in vivo, efforts are currently being under taken to identifynew, native proteins which are involved in synaptic machinery and celladhesion. We describe herein the identification of novel polypeptideswhich have homology to densin, designated herein as PRO239 polypeptides.

30. PRO257

Ebnerin is a cell surface protein associated with von Ebner glands inmammals. Efforts are being undertaken by both industry and academia toidentify new, native cell surface receptor proteins and specificallythose which possess sequence homology to cell surface proteins such asebnerin. Many of these efforts are focused on the screening of mammalianrecombinant DNA libraries to identify the coding sequences for novelreceptor proteins. We herein describe the identification of novelpolypeptides having significant homology to the von Ebner'sgland-associated protein ebnerin, designated herein as PRO257polypeptides.

31. PRO260

Fucosidases are enzymes that remove fucose residues from fucosecontaining proteoglycans. In some pathological conditions, such ascancer, rheumatoid arthritis, and diabetes, there is an abnormalfucosylation of serum proteins. Therefore, fucosidases, and proteinshaving homology to fucosidase, are of importance to the study andabrogation of these conditions. In particular, proteins having homologyto the alpha-1-fucosidase precursor are of interest. Fucosidases andfucosidase inhibitors are further described in U.S. Pat. Nos. 5,637,490,5,382,709, 5,240,707, 5,153,325, 5,100,797, 5,096,909 and 5,017,704.Studies are also reported in Valk, et al., J. Virol., 71(9):6796 (1997),Aktogu, et al., Monaldi. Arch. Chest Dis. (Italy), 52(2);54 (1997). andFocarelli, et al., Biochem. Biophys. Res. Commun. (U.S.), 234(1):54(1997).

Efforts are being undertaken by both industry and academia to identifynew, native secreted and membrane-bound receptor proteins. Of particularinterest are proteins having homology to the alpha-1-fucosidaseprecursor. Many efforts are focused on the screening of mammalianrecombinant DNA libraries to identify the coding sequences for novelsecreted and membrane-bound receptor proteins. Examples of screeningmethods and techniques are described in the literature [see, forexample, Klein et al., Proc. Natl. Acad. Sci., 93:7108-7113 (1996); U.S.Pat. No. 5,536,637)].

We herein describe the identification and characterization of novelpolypeptides having homology to fucosidases, designated herein as PRO260polypeptides.

32. PRO263

CD44 is a cell surface adhesion molecule involved in cell-cell andcell-matrix interactions. Hyaluronic acid, a component of theextracellular matrix is a major ligand. Other ligands include collagen,fibronectin, laminin, chrondroitin sulfate, mucosal addressin, serglycinand osteoponin. CD44 is also important in regulating cell traffic, lymphnode homing, transmission of growth signals, and presentation ofchemokines and growth factors to traveling cells. CD44 surface proteinsare associated with metastatic tumors and CD44 has been used as a markerfor HIV infection. Certain splice variants are associated withmetastasis and poor prognosis of cancer patients. Therefore, moleculeshaving homology with CD44 are of particular interest, as their homologyindicates that they may have functions related to those functions ofCD44. CD44 is further described in U.S. Pat. Nos. 5,506,119, 5,504,194and 5,108,904; Gerberick, et al., Toxicol. Appl. Pharmacol., 146(1):1(1997); Wittig, et al., Immunol. Letters (Netherlands), 57(1-3):217(1997); and Oliveira and Odell, Oral Oncol. (England), 33(4):260 (1997).

Efforts are being undertaken by both industry and academia to identifynew, native secreted and membrane-bound receptor proteins, particularlytransmembrane proteins with homology to CD44 antigen. Many efforts arefocused on the screening of mammalian recombinant DNA libraries toidentify the coding sequences for novel secreted and membrane-boundreceptor proteins. Examples of screening methods and techniques aredescribed in the literature [see, for example, Klein et al., Proc. Natl.Acad. Sci., 93:7108-7113 (1996); U.S. Pat. No. 5,536,637)].

We herein describe the identification and characterization of novelpolypeptides having homology to CD44 antigen, designated herein asPRO263 polypeptides.

33. PRO270

Thioredoxins effect reduction-oxidation (redox) state. Many diseases arepotentially related to redox state and reactive oxygen species may playa role in many important biological processes. The transcriptionfactors, NF-kappa B and AP-1, are regulated by redox state and are knownto affect the expression of a large variety of genes thought to beinvolved in the pathogenesis of AIDS, cancer, atherosclerosis anddiabetic complications. Such proteins may also play a role in cellularantioxidant defense, and in pathological conditions involving oxidativestress such as stroke and inflammation in addition to having a role inapoptosis. Therefore, thioredoxins, and proteins having homologythereto, are of interest to the scientific and medical communities.

We herein describe the identification and characterization of novelpolypeptides having homology to thioredoxin, designated herein as PRO270polypeptides.

34. PRO271

The proteoglycan link protein is a protein which is intimatelyassociated with various extracellular matrix proteins and morespecifically with proteins such as collagen. For example, one primarycomponent of collagen is a large proteoglycan called aggrecan. Thismolecule is retained by binding to the glycosaminoglycan hyaluronanthrough the amino terminal G1 globular domain of the core protein. Thisbinding is stabilized by the proteoglycan link protein which is aprotein that is also associated with other tissues containing hyaluronanbinding proteoglycans such as versican.

Link protein has been identified as a potential target for autoimmuneantibodies in individuals who suffer from juvenile rheumatoid arthritis(see Guerassimov et al., J. Rheumatology 24(5):959-964 (1997)). As such,there is strong interest in identifying novel proteins having homologyto link protein. We herein describe the identification andcharacterization of novel polypeptides having such homology, designatedherein as PRO271 polypeptides.

35. PRO272

Reticulocalbin is an endoplasmic reticular protein which may be involvedin protein transport and luminal protein processing. Reticulocalbinresides in the lumen of the endoplasmic rerticulum, is known to bindcalcium, and may be involved in a luminal retention mechanism of theendoplasmic reticulum. It contains six domains of the EF-hand motifassociated with high affinity calcium binding. We describe herein theidentification and characterization of a novel polypeptide which hashomology to the reticulocalbin protein, designated herein as PRO272.

36. PRO294

Collagen, a naturally occurring protein, finds wide application inindustry. Chemically hydrolyzed natural collagen can be denatured andrenatured by heating and cooling to produce gelatin, which is used inphotographic and medical, among other applications. Collagen hasimportant properties such as the ability to form interchain aggregateshaving a conformation designated as a triple helix. We herein describethe identification and characterization of a novel polypeptide which hashomology to portions of the collagen molecule, designated herein asPRO294.

37. PRO295

The integrins comprise a supergene family of cell-surface glycoproteinreceptors that promote cellular adhesion. Each cell has numerousreceptors that define its cell adhesive capabilities. Integrins areinvolved in a wide variety of interaction between cells and other cellsor matrix components. The integrins are of particular importance inregulating movement and function of immune system cells The plateletIIb/IIIA integrin complex is of particular importance in regulatingplatelet aggregation. A member of the integrin family, integrin β-6, isexpressed on epithelial cells and modulates epithelial inflammation.Another integrin, leucocyte-associated antigen-1 (LFA-1) is important inthe adhesion of lymphocytes during an immune response. The integrins areexpressed as heterodimers of non-covalently associated alpha and betasubunits. Given the physiological importance of cell adhesion mechanismsin vivo, efforts are currently being under taken to identify new, nativeproteins which are involved in cell adhesion. We describe herein theidentification and characterization of a novel polypeptide which hashomology to integrin, designated herein as PRO295.

38. PRO293

Protein-protein interactions include receptor and antigen complexes andsignaling mechanisms. As more is known about the structural andfunctional mechanisms underlying protein-protein interactions,protein-protein interactions can be more easily manipulated to regulatethe particular result of the protein-protein interaction. Thus, theunderlying mechanisms of protein-protein interactions are of interest tothe scientific and medical community.

All proteins containing leucine-rich repeats are thought to be involvedin protein-protein interactions. Leucine-rich repeats are short sequencemotifs present in a number of proteins with diverse functions andcellular locations. The crystal structure of ribonuclease inhibitorprotein has revealed that leucine-rich repeats correspond to beta-alphastructural units. These units are arranged so that they form a parallelbeta-sheet with one surface exposed to solvent, so that the proteinacquires an unusual, nonglubular shape. These two features have beenindicated as responsible for the protein-binding functions of proteinscontaining leucine-rich repeats. See, Kobe and Deisenhofer, TrendsBiochem. Sci., 19(10):415-421 (October 1994).

A study has been reported on leucine-rich proteoglycans which serve astissue organizers, orienting and ordering collagen fibrils duringontogeny and are involved in pathological processes such as woundhealing, tissue repair, and tumor stroma formation. Iozzo, R. V., Crit.Rev. Biochem. Mol. Biol., 32(2):141-174 (1997). Others studiesimplicating leucine rich proteins in wound healing and tissue repair areDe La Salle, C., et al., Vouv. Rev. Fr. Hematol. (Germany),37(4):215-222 (1995), reporting mutations in the leucine rich motif in acomplex associated with the bleeding disorder Bernard-Soulier syndromeand Chlemetson, K. J., Thromb. Haemost. (Germany), 74(1): 111-116 (July1995), reporting that platelets have leucine rich repeats. Anotherprotein of particular interest which has been reported to haveleucine-rich repeats is the SLIT protein which has been reported to beuseful in treating neuro-degenerative diseases such as Alzheimer'sdisease, nerve damage such as in Parkinson's disease, and for diagnosisof cancer, see, Artavanistsakonas, S. and Rothberg, J. M., WO9210518-A1by Yale University. Other studies reporting on the biological functionsof proteins having leucine-rich repeats include: Tayar, N., et al., Mol.Cell Endocrinol., (Ireland), 125(1-2):65-70 (December 1996)(gonadotropin receptor involvement); Miura, Y., et al., Nippon Rinsho(Japan), 54(7):1784-1789 (July 1996) (apoptosis involvement); Harris, P.C., et al., J. Am. Soc. Nephrol., 6(4):1125-1133 (October 1995) (kidneydisease involvement); and Ruoslahti, E. I., et al., WO9110727-A by LaJolla Cancer Research Foundation (decorin binding to transforming growthfactorβ involvement for treatment for cancer, wound healing andscarring).

Efforts are therefore being undertaken by both industry and academia toidentify new proteins having leucine rich repeats to better understandprotein-protein interactions. Of particular interest are those proteinshaving leucine rich repeats and homology to known neuronal leucine richrepeat proteins. Many efforts are focused on the screening of mammalianrecombinant DNA libraries to identify the coding sequences for novelsecreted and membrane-bound proteins having leucine rich repeats.Examples of screening methods and techniques are described in theliterature [see, for example, Klein et al., Proc. Natl. Acad. Sci.,93:7108-7113 (1996); U.S. Pat. No. 5,536,637)].

We describe herein the identification and characterization of a novelpolypeptide which has homology to leucine rich repeat proteins,designated herein as PRO293.

39. PRO247

Protein-protein interactions include receptor and antigen complexes andsignaling mechanisms. As more is known about the structural andfunctional mechanisms underlying protein-protein interactions,protein-protein interactions can be more easily manipulated to regulatethe particular result of the protein-protein interaction. Thus, theunderlying mechanisms of protein-protein interactions are of interest tothe scientific and medical community.

All proteins containing leucine-rich repeats are thought to be involvedin protein-protein interactions. Leucine-rich repeats are short sequencemotifs present in a number of proteins with diverse functions andcellular locations. The crystal structure of ribonuclease inhibitorprotein has revealed that leucine-rich repeats correspond to beta-alphastructural units. These units are arranged so that they form a parallelbeta-sheet with one surface exposed to solvent, so that the proteinacquires an unusual, nonglubular shape. These two features have beenindicated as responsible for the protein-binding functions of proteinscontaining leucine-rich repeats. See, Kobe and Deisenhofer, TrendsBiochem. Sci., 19(10):415-421 (October 1994).

A study has been reported on leucine-rich proteoglycans which serve astissue organizers, orienting and ordering collagen fibrils duringontogeny and are involved in pathological processes such as woundhealing, tissue repair, and tumor stroma formation. Iozzo, R. V., Crit.Rev. Biochem. Mol. Biol., 32(2):141-174 (1997). Others studiesimplicating leucine rich proteins in wound healing and tissue repair areDe La Salle, C., et al., Vouv. Rev. Fr. Hematol. (Germany),37(4):215-222 (1995), reporting mutations in the leucine rich motif in acomplex associated with the bleeding disorder Bernard-Soulier syndromeand Chlemetson, K. J., Thromb. Haemost. (Germany), 74(1):111-116 (July1995), reporting that platelets have leucine rich repeats. Anotherprotein of particular interest which has been reported to haveleucine-rich repeats is the SLIT protein which has been reported to beuseful in treating neuro-degenerative diseases such as Alzheimer'sdisease, nerve damage such as in Parkinson's disease, and for diagnosisof cancer, see, Artavanistsakonas, S. and Rothberg, J. M., WO9210518-A1by Yale University. Other studies reporting on the biological functionsof proteins having leucine-rich repeats include: Tayar, N., et al., Mol.Cell Endocrinol., (Ireland), 125(1-2):65-70 (December 1996)(gonadotropin receptor involvement); Miura, Y., et al., Nippon Rinsho(Japan), 54(7): 1784-1789 (July 1996) (apoptosis involvement); Harris,P. C., et al., J. Am. Soc. Nephrol., 6(4):1125-1133 (October 1995)(kidney disease involvement); and Ruoslahti, E. I., et al., WO9110727-Aby La Jolla Cancer Research Foundation (decorin binding to transforminggrowth factorβ involvement for treatment for cancer, wound healing andscarring).

Densin is a glycoprotein which has been isolated from the brain whichhas all the hallmarks of an adhesion molecule. It is highly concentratedat synaptic sites in the brain and is expressed prominently in dendriticprocesses in developing neurons. Densin has been characterized as amember of the O-linked sialoglycoproteins. Densin has relevance tomedically important processes such as regeneration. Given thephysiological importance of synaptic processes and cell adhesionmechanisms in vivo, efforts are currently being under taken to identifynew, native proteins which are involved in synaptic machinery and celladhesion. Densin is further described in Kennedy, M. B, Trends Neurosci.(England), 20(6):264 (1997) and Apperson, et al., J. Neurosci.,16(21):6839 (1996).

Efforts are therefore being undertaken by both industry and academia toidentify new proteins having leucine rich repeats to better understandprotein-protein interactions. Of particular interest are those proteinshaving leucine rich repeats and homology to known proteins havingleucine rich repeats such as KIAA0231 and densin. Many efforts arefocused on the screening of mammalian recombinant DNA libraries toidentify the coding sequences for novel secreted and membrane-boundproteins having leucine rich repeats. Examples of screening methods andtechniques are described in the literature [see, for example, Klein etal., Proc. Natl. Acad. Sci., 93:7108-7113 (1996); U.S. Pat. No.5,536,637)].

We describe herein the identification and characterization of a novelpolypeptide which has homology to leucine rich repeat proteins,designated herein as PRO247.

40. PRO302, PRO303, PRO304, PRO307 and PRO343

Proteases are enzymatic proteins which are involved in a large number ofvery important biological processes in mammalian and non-mammalianorganisms. Numerous different protease enzymes from a variety ofdifferent mammalian and non-mammalian organisms have been bothidentified and characterized. The mammalian protease enzymes playimportant roles in many different biological processes including, forexample, protein digestion, activation, inactivation, or modulation ofpeptide hormone activity, and alteration of the physical properties ofproteins and enzymes.

In light of the important physiological roles played by proteaseenzymes, efforts are currently being undertaken by both industry andacademia to identify new, native protease homologs. Many of theseefforts are focused on the screening of mammalian recombinant DNAlibraries to identify the coding sequences for novel secreted andmembrane-bound receptor proteins. Examples of screening methods andtechniques are described in the literature [see, for example, Klein etal., Proc. Natl. Acad. Sci., 93:7108-7113 (1996); U.S. Pat. No.5,536,637)]. We herein describe the identification of novel polypeptideshaving homology to various protease enzymes, designated herein asPRO302, PRO303, PRO304, PRO307 and PRO343 polypeptides.

41. PRO328

The GLIP protein family has been characterized as comprising zinc-fingerproteins which play important roles in embryogenesis. These proteins mayfunction as transcriptional regulatory proteins and are known to beamplified in a subset of human tumors. Glioma pathogenesis protein isstructurally related to a group of plant pathogenesis-related proteins.It is highly expressed in glioblastoma. See U.S. Pat. No. 5,582,981(issued Dec. 10, 1996) and U.S. Pat. No. 5,322,801 (issued Jun. 21,1996), Ellington, A. D. et al., Nature, 346:818 (1990), Grindley, J. C.et al., Dev. Biol., 188(2):337 (1997), Marine, J. C. et al., Mech. Dev.,63(2):211 (1997). The CRISP or cysteine rich secretory protein familyare a group of proteins which are also structurally related to a groupof plant pathogenesis proteins. [Schwidetzky, U., Biochem. J., 321:325(1997), Pfisterer, P., Mol. Cell Biol., 16(11):6160 (1996), Kratzschmar,J. Eur. J. Biochem., 236(3):827 (1996)]. We describe herein theidentification of a novel polypeptide which has homology to GLIP andCRISP, designated herein as PRO328 polypeptides.

42. PRO335, PRO331 and PRO326

Protein-protein interactions include receptor and antigen complexes andsignaling mechanisms. As more is known about the structural andfunctional mechanisms underlying protein-protein interactions,protein-protein interactions can be more easily manipulated to regulatethe particular result of the protein-protein interaction. Thus, theunderlying mechanisms of protein-protein interactions are of interest tothe scientific and medical community.

All proteins containing leucine-rich repeats are thought to be involvedin protein-protein interactions. Leucine-rich repeats are short sequencemotifs present in a number of proteins with diverse functions andcellular locations. The crystal structure of ribonuclease inhibitorprotein has revealed that leucine-rich repeats correspond to beta-alphastructural units. These units are arranged so that they form a parallelbeta-sheet with one surface exposed to solvent, so that the proteinacquires an unusual, nonglubular shape. These two features have beenindicated as responsible for the protein-binding functions of proteinscontaining leucine-rich repeats. See, Kobe and Deisenhofer, TrendsBiochem. Sci., 19(10):415421 (October 1994).

A study has been reported on leucine-rich proteoglycans which serve astissue organizers, orienting and ordering collagen fibrils duringontogeny and are involved in pathological processes such as woundhealing, tissue repair, and tumor stroma formation. Iozzo, R. V., Crit.Rev. Biochem. Mol. Biol., 32(2):141-174 (1997). Others studiesimplicating leucine rich proteins in wound healing and tissue repair areDe La Salle, C., et al., Vouv. Rev. Fr. Hematol. (Germany),37(4):215-222 (1995), reporting mutations in the leucine rich motif in acomplex associated with the bleeding disorder Bernard-Soulier syndrome,Chlemetson, K. J., Thromb. Haemost. (Germany), 74(1): 111-116 (July1995), reporting that platelets have leucine rich repeats and Ruoslahti,E. I., et al., WO9110727-A by La Jolla Cancer Research Foundationreporting that decorin binding to transforming growth factors hasinvolvement in a treatment for cancer, wound healing and scarring.Related by function to this group of proteins is the insulin like growthfactor (IGF), in that it is useful in wound-healing and associatedtherapies concerned with re-growth of tissue, such as connective tissue,skin and bone; in promoting body growth in humans and animals; and instimulating other growth-related processes. The acid labile subunit ofIGF (ALS) is also of interest in that it increases the half-life of IGFand is part of the IGF complex in vivo.

Another protein which has been reported to have leucine-rich repeats isthe SLIT protein which has been reported to be useful in treatingneuro-degenerative diseases such as Alzheimer's disease, nerve damagesuch as in Parkinson's disease, and for diagnosis of cancer, see,Artavanistsakonas, S. and Rothberg, J. M., WO9210518-A1 by YaleUniversity. Of particular interest is LIG-1, a membrane glycoproteinthat is expressed specifically in glial cells in the mouse brain, andhas leucine rich repeats and immunoglobulin-like domains. Suzuki, etal., J. Biol. Chem. (U.S.), 271(37):22522 (1996). Other studiesreporting on the biological functions of proteins having leucine richrepeats include: Tayar, N., et al., Mol. Cell Endocrinol., (Ireland),125(1-2):65-70 (December 1996) (gonadotropin receptor involvement);Miura, Y., et al., Nippon Rinsho (Japan), 54(7):1784-1789 (July 1996)(apoptosis involvement); Harris, P. C., et al., J. Am. Soc. Nephrol.,6(4):1125-1133 (October 1995) (kidney disease involvement).

Efforts are therefore being undertaken by both industry and academia toidentify new proteins having leucine rich repeats to better understandprotein-protein interactions. Of particular interest are those proteinshaving leucine rich repeats and homology to known proteins havingleucine rich repeats such as LIG-1, ALS and decorin. Many efforts arefocused on the screening of mammalian recombinant DNA libraries toidentify the coding sequences for novel secreted and membrane-boundproteins having leucine rich repeats. Examples of screening methods andtechniques are described in the literature [see, for example, Klein etal., Proc. Natl Acad. Sci., 93:7108-7113 (1996); U.S. Pat. No.5,536,637)].

We describe herein the identification and characterization of novelpolypeptides which have homology to proteins of the leucine rich repeatsuperfamily, designated herein as PRO335, PRO331 and PRO326polypeptides.

43. PRO332

Secreted proteins comprising a repeat characterized by an arrangement ofconserved leucine residues (leucine-rich repeat motif) have diversebiological roles. Certain proteoglycans, such as biglycan, fibromodulinand decorin, are, for example, characterized by the presence of aleucine-rich repeat of about 24 amino acids [Ruoslahti, Ann. Rev. Cell.Biol. 4 229-255 (1988); Oldberg et al., EMBO J. 8, 2601-2604 (1989)]. Ingeneral, proteoglycans are believed to play a role in regulatingextracellular matrix, cartilage or bone function. The proteoglycandecorin binds to collagen type I and II and affects the rate of fibrilformation. Fibromodulin also binds collagen and delays fibril formation.Both fibromodulin and decorin inhibit the activity of transforminggrowth factor beta (TGF-β) (U.S. Pat. No. 5,583,103 issued Dec. 10,1996). TGF-β is known to play a key role in the induction ofextracellular matrix and has been implicated in the development offibrotic diseases, such as cancer and glomerulonephritis. Accordingly,proteoglycans have been proposed for the treatment of fibrotic cancer,based upon their ability to inhibit TGF-β's growth stimulating activityon the cancer cell. Proteoglycans have also been described aspotentially useful in the treatment of other proliferative pathologies,including rheumatoid arthritis, arteriosclerosis, adult respiratorydistress syndrome, cirrhosis of the liver, fibrosis of the lungs,post-myocardial infarction, cardiac fibrosis, post-angioplastyrestenosis, renal interstitial fibrosis and certain dermal fibroticconditions, such as keloids and scarring, which might result from burninjuries, other invasive skin injuries, or cosmetic or reconstructivesurgery (U.S. Pat. No. 5,654,270, issued Aug. 5, 1997).

We describe herein the identification and characterization of novelpolypeptides which have homology to proteins of the leucine rich repeatsuperfamily, designated herein as PRO332 polypeptides.

44. PRO334

Microfibril bundles and proteins found in association with thesebundles, particularly attachment molecules, are of interest in the fieldof dermatology, particularly in the study of skin which has been damagedfrom aging, injuries or the sun. Fibrillin microfibrils define thecontinuous elastic network of skin, and are present in dermis asmicrofibril bundles devoid of measurable elastin extending from thedermal-epithelial junction and as components of the thick elastic fibrespresent in the deep reticular dermis. Moreover, Marfan syndrome has beenlinked to mutations which interfere with multimerization of fibrillinmonomers or other connective tissue elements.

Fibulin-1 is a modular glycoprotein with amino-terminalanaphlatoxin-like modules followed by nine epidermal growth factor(EGF)-like modules and, depending on alternative splicing, four possiblecarboxyl termini. Fibulin-2 is a novel extracellular matrix proteinfrequently found in close association with microfibrils containingeither fibronectin or fibrillin. Thus, fibrillin, fibulin, and moleculesrelated thereto are of interest, particularly for the use of preventingskin from being damaged from aging, injuries or the sun, or forrestoring skin damaged from same. Moreover, these molecules aregenerally of interest in the study of connective tissue and attachmentmolecules and related mechanisms. Fibrillin, fibulin and relatedmolecules are further described in Adams, et al., J. Mol. Biol.,272(2):226-36 (1997); Kielty and Shuttleworth, Microsc. Res. Tech.,38(4): 413-27 (1997); and Child, J. Card. Surg. 12(2Supp.):131-5 (1997).

Currently, efforts are being undertaken by both industry and academia toidentify new, native secreted and membrane-bound receptor proteins,particularly secreted proteins which have homology to fibulin andfibrillin. Many efforts are focused on the screening of mammalianrecombinant DNA libraries to identify the coding sequences for novelsecreted and membrane-bound receptor proteins. Examples of screeningmethods and techniques are described in the literature [see, forexample, Klein et al., Proc. Natl. Acad. Sci., 93:7108-7113 (1996); U.S.Pat. No. 5,536,637)].

We herein describe the identification and characterization of novelpolypeptides having homology to fibulin and fibrillin, designated hereinas PRO334 polypeptides.

45. PRO346

The widespread occurrence of cancer has prompted the devotion ofconsiderable resources and discovering new treatments of treatment. Oneparticular method involves the creation of tumor or cancer specificmonoclonal antibodies (mAbs) which are specific to tumor antigens. SuchmAbs, which can distinguish between normal and cancerous cells areuseful in the diagnosis, prognosis and treatment of the disease.Particular antigens are known to be associated with neoplastic diseases,such as colorectal and breast cancer. Since colon cancer is a widespreaddisease, early diagnosis and treatment is an important medical goal.Diagnosis and treatment of cancer can be implemented using monoclonalantibodies (mAbs) specific therefore having fluorescent, nuclearmagnetic or radioactive tags. Radioactive genes, toxins and/or drugtagged mAbs can be used for treatment in situ with minimal patientdescription.

Carcinoembryonic antigen (CEA) is a glycoprotein found in human coloncancer and the digestive organs of a 2-6 month human embryos. CEA is aknown human tumor marker and is widely used in the diagnosis ofneoplastic diseases, such as colon cancer. For example, when the serumlevels of CEA are elevated in a patient, a drop of CEA levels aftersurgery would indicate the tumor resection was successful. On the otherhand, a subsequent rise in serum CEA levels after surgery would indicatethat metastases of the original tumor may have formed or that newprimary tumors may have appeared. CEA may also be a target for mAb,antisense nucleotides

46. PRO268

Protein disulfide isomerase is an enzymatic protein which is involved inthe promotion of correct refolding of proteins through the establishmentof correct disulfide bond formation. Protein disulfide isomerase wasinitially identified based upon its ability to catalyze the renaturationof reduced denatured RNAse (Goldberger et al., J. Biol. Chem.239:1406-1410 (1964) and Epstein et al., Cold Spring Harbor Symp. Quant.Biol. 28:439-449 (1963)). Protein disulfide isomerase has been shown tobe a resident enzyme of the endoplasmic reticulum which is retained inthe endoplasmic reticulum via a -KDEL or -HDEL amino acid sequence atits C-terminus.

Given the importance of disulfide bond-forming enzymes and theirpotential uses in a number of different applications, for example inincreasing the yield of correct refolding of recombinantly producedproteins, efforts are currently being undertaken by both industry andacademia to identify new, native proteins having homology to proteindisulfide isomerase. Many of these efforts are focused on the screeningof mammalian recombinant DNA libraries to identify the coding sequencesfor novel protein disulfide isomerase homologs. We herein describe anovel polypeptide having homology to protein disulfide isomerase,designated herein as PRO268.

47. PRO330

Prolyl 4-hydroxylase is an enzyme which functions topost-translationally hydroxylate proline residues at the Y position ofthe amino acid sequence Gly-X-Y, which is a repeating three amino acidsequence found in both collagen and procollagen. Hydroxylation ofproline residues at the Y position of the Gly-X-Y amino acid triplet toform 4-hydroxyproline residues at those positions is required beforenewly synthesized collagen polypeptide chains may fold into their properthree-dimensional triple-helical conformation. If hydroxylation does notoccur, synthesized collagen polypeptides remain non-helical, are poorlysecreted by cells and cannot assemble into stable functional collagenfibrils. Vuorio et al., Proc. Natl. Acad. Sci. USA 89:7467-7470 (1992).Prolyl 4-hydroxylase is comprised of at least two different polypeptidesubunits, alpha and beta.

Efforts are being undertaken by both industry and academia to identifynew, native secreted and membrane-bound receptor proteins. Many effortsare focused on the screening of mammalian recombinant DNA libraries toidentify the coding sequences for novel secreted and membrane-boundreceptor proteins. Examples of screening methods and techniques aredescribed in the literature [see, for example, Klein et al., Proc. Natl.Acad. Sci., 93:7108-7113 (1996); U.S. Pat. No. 5,536,637)]. Based uponthese efforts, Applicants have herein identified and describe a novelpolypeptide having homology to the alpha subunit of prolyl4-hydroxylase, designated herein as PRO330.

48. PRO339 and PRO310

Fringe is a protein which specifically blocks serrate-mediatedactivation of notch in the dorsal compartment of the Drosophila wingimaginal disc. Fleming, et al., Development, 124(15):2973-81 (1997).Therefore, fringe is of interest for both its role in development aswell as its ability to regulate serrate, particularly serrate'ssignaling abilities. Also of interest are novel polypeptides which mayhave a role in development and/or the regulation of serrate-likemolecules. Of particular interest are novel polypeptides having homologyto fringe as identified and described herein, designated herein asPRO339 and PRO310 polypeptides.

49. PRO244

Lectins are a class of proteins comprising a region that bindscarbohydrates specifically and non-covalently. Numerous lectins havebeen identified in higher animals, both membrane-bound and soluble, andhave been implicated in a variety of cell-recognition phenomena andtumor metastasis.

Most lectins can be classified as either C-type (calcium-dependent) orS-type (thiol-dependent).

Lectins are thought to play a role in regulating cellular events thatare initiated at the level of the plasma membrane. For example, plasmamembrane associated molecules are involved in the activation of varioussubsets of lymphoid cells, e.g. T-lymphocytes, and it is known that cellsurface molecules are responsible for activation of these cells andconsequently their response during an immune reaction.

A particular group of cell adhesion molecules, selecting, belong in thesuperfamily of C-type lectins. This group includes L-selectin(peripheral lymph node homing receptor (pnHR), LEC-CAM-1, LAM-1,gp90^(MEL), gp₁₀₀ ^(MEL), gp110^(MEL), MEL-14 antigen, Leu-8 antigen,TQ-1 antigen, DREG antigen), E-selectin (LEC-CAM-2, LECAM-2, ELAM-1),and P-selectin (LEC-CAM-3, LECAM-3, GMP-140, PADGEM). The structure ofselectins consists of a C-type lectin (carbohydrate binding) domain, anepidermal growth factor-like (EGF-like) motif, and variable numbers ofcomplement regulatory (CR) motifs. Selectins are associated withleukocyte adhesion, e.g. the attachment of neutrophils to venularendothelial cells adjacent to inflammation (E-selectin), or with thetrafficking of lymphocytes from blood to secondary lymphoid organs, e.g.lymph nodes and Peyer's patches (L-selectin).

Another exemplary lectin is the cell-associated macrophage antigen,Mac-2 that is believed to be involved in cell adhesion and immuneresponses. Macrophages also express a lectin that recognizes Tn Ag, ahuman carcinoma-associated epitope.

Another C-type lectin is CD95 (Fas antigen/APO-1) that is an importantmediator of immunologically relevant regulated or programmed cell death(apoptosis). “Apoptosis” is a non-necrotic cell death that takes placein metazoan animal cells following activation of an intrinsic cellsuicide program. The cloning of Fas antigen is described in PCTpublication WO 91/10448, and European patent application EP510691. Themature Fas molecule consists of 319 amino acids of which 157 areextracellular, 17 constitute the transmembrane domain, and 145 areintracellular. Increased levels of Fas expression at T cell surface havebeen associated with tumor cells and HIV-infected cells. Ligation ofCD95 triggers apoptosis in the presence of interleukin-1 (IL-2).

C-type lectins also include receptors for oxidized low-densitylipoprotein (LDL). This suggests a possible role in the pathogenesis ofatherosclerosis.

We herein describe the identification and characterization of novelpolypeptides having homology to C-type lectins, designated herein asPRO244 polypeptides.

SUMMARY OF THE INVENTION

1. PRO211 and PRO217

Applicants have identified cDNA clones that encode novel polypeptideshaving homology to EGF, designated in the present application as“PRO211” and “PRO217” polypeptides.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO211 or PRO217 polypeptide. In oneaspect, the isolated nucleic acid comprises DNA encoding EGF-likehomologue PRO211 and PRO217 polypeptides of FIG. 2 (SEQ ID NO:2) and/or4 (SEQ ID NO:4) indicated in FIG. 1 (SEQ ID NO1) and/or FIG. 3 (SEQ IDNO:3), respectively, or is complementary to such encoding nucleic acidsequence, and remains stably bound to it under at least moderate, andoptionally, under high stringency conditions.

In another embodiment, the invention provides isolated PRO211 and PRO217EGF-like homologue PRO211 and PRO217 polypeptides. In particular, theinvention provides isolated native sequence PRO211 and PRO217 EGF-likehomologue polypeptides, which in one embodiment, includes an amino acidsequence comprising residues: 1 to 353 of FIG. 2 (SEQ ID NO:2) or (2) 1to 379 of FIG. 4 (SEQ ID NO:4).

2. PRO230

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO230”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO230 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO230 polypeptidehaving amino acid residues 1 through 467 of FIG. 6 (SEQ ID NO:12), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In another embodiment, the invention providesisolated PRO230 polypeptide. In particular, the invention providesisolated native sequence PRO230 polypeptide, which in one embodiment,includes an amino acid sequence comprising residues 1 through 467 ofFIG. 6 (SEQ ID NO:12).

In another embodiment, the invention provides an expressed sequence tag(EST) comprising the nucleotide sequence of SEQ ID NO:13 (FIG. 7) whichis herein designated as DNA20088.

3. PRO232

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO232”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO232 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO232 polypeptidehaving amino acid residues 1 to 114 of FIG. 9 (SEQ ID NO:18), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO232polypeptide. In particular, the invention provides isolated nativesequence PRO232 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 114 of FIG. 9 (SEQ ID NO:18).

4. PRO187

Applicants have identified a cDNA clone that encodes a novelpolypeptide, designated in the present application as “PRO187”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO187 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO187 polypeptideof FIG. 11 (SEQ ID NO:23), or is complementary to such encoding nucleicacid sequence, and remains stably bound to it under at least moderate,and optionally, under high stringency conditions. In another aspect, theinvention provides a nucleic acid comprising the coding sequence of FIG.10 (SEQ ID NO:22) or its complement. In another aspect, the inventionprovides a nucleic acid of the full length protein of cloneDNA27864-1155, deposited with the ATCC under accession number ATCC209375, alternatively the coding sequence of clone DNA27864-1155,deposited under accession number ATCC 209375.

In yet another embodiment, the invention provides isolated PRO187polypeptide. In particular, the invention provides isolated nativesequence PRO187 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 205 of FIG. 11 (SEQ ID NO:23).Alternatively, the invention provides a polypeptide encoded by thenucleic acid deposited under accession number ATCC 209375.

5. PRO265

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO265”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO265 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO265 polypeptidehaving amino acid residues 1 to 660 of FIG. 13 (SEQ ID NO:28), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO265polypeptide. In particular, the invention provides isolated nativesequence PRO265 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 660 of FIG. 13 (SEQ ID NO:28). Anadditional embodiment of the present invention is directed to anisolated extracellular domain of a PRO265 polypeptide.

6. PRO219

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO219”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO219 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO219 polypeptidehaving amino acid residues 1 to 915 of FIG. 15 (SEQ ID NO:34), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO219polypeptide. In particular, the invention provides isolated nativesequence PRO219 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 915 of FIG. 15 (SEQ ID NO:34).

7. PRO246

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO246”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO246 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO246 polypeptidehaving amino acid residues 1 to 390 of FIG. 17 (SEQ ID NO:39), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO246polypeptide. In particular, the invention provides isolated nativesequence PRO246 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 390 of FIG. 17 (SEQ ID NO:39). Anadditional embodiment of the present invention is directed to anisolated extracellular domain of a PRO246 polypeptide.

8. PRO228

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to CD97, EMR1 and latrophilin, wherein the polypeptideis designated in the present application as “PRO228”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO228 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO228 polypeptidehaving amino acid residues 1 to 690 of FIG. 19 (SEQ ID NO:49), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO228polypeptide. In particular, the invention provides isolated nativesequence PRO228 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 690 of FIG. 19 (SEQ ID NO:49). Anadditional embodiment of the present invention is directed to anisolated extracellular domain of a PRO228 polypeptide.

In another embodiment, the invention provides an expressed sequence tag(EST) comprising the nucleotide sequence of SEQ ID NO:50, designatedherein as DNA21951.

9. PRO533

Applicants have identified a cDNA clone (DNA49435-1219) that encodes anovel polypeptide, designated in the present application as PRO533.

In one embodiment, the invention provides an isolated nucleic acidmolecule having at least about 80% sequence identity to (a) a DNAmolecule encoding a PRO533 polypeptide comprising the sequence of aminoacids 23 to 216 of FIG. 22 (SEQ ID NO:59), or (b) the complement of theDNA molecule of (a). The sequence identity preferably is about 85%, morepreferably about 90%, most preferably about 95%. In one aspect, theisolated nucleic acid has at least about 80%, preferably at least about85%, more preferably at least about 90%, and most preferably at leastabout 95% sequence identity with a polypeptide having amino acidresidues 23 to 216 of FIG. 22 (SEQ ID NO:59). Preferably, the highestdegree of sequence identity occurs within the secreted portion (aminoacids 23 to 216 of FIG. 22, SEQ ID NO:59). In a further embodiment, theisolated nucleic acid molecule comprises DNA encoding a PRO533polypeptide having amino acid residues 1 to 216 of FIG. 22 (SEQ IDNO:59), or is complementary to such encoding nucleic acid sequence, andremains stably bound to it under at least moderate, and optionally,under high stringency conditions. In another aspect, the inventionprovides a nucleic acid of the full length protein of cloneDNA49435-1219, deposited with the ATCC under accession number ATCC209480.

In yet another embodiment, the invention provides isolated PRO533polypeptide. In particular, the invention provides isolated nativesequence PRO533 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 23 to 216 of FIG. 22 (SEQ ID NO:59).Native PRO533 polypeptides with or without the native signal sequence(amino acids 1 to 22 in FIG. 22 (SEQ ID NO:59)), and with or without theinitiating methionine are specifically included. Alternatively, theinvention provides a PRO533 polypeptide encoded by the nucleic aciddeposited under accession number ATCC 209480.

10. PRO245

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO245”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO245 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO245 polypeptidehaving amino acid residues 1 to 312 of FIG. 24 (SEQ ID NO:64), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO245polypeptide. In particular, the invention provides isolated nativesequence PRO245 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 312 of FIG. 24 (SEQ ID NO:64).

11. PRO220, PRO221 and PRO227

Applicants have identified cDNA clones that each encode novelpolypeptides, all having leucine rich repeats. These polypeptides aredesignated in the present application as PRO220, PRO221 and PRO227.

In one embodiment, the invention provides isolated nucleic acidmolecules comprising DNA respectively encoding PRO220, PRO221 andPRO227, respectively. In one aspect, provided herein is an isolatednucleic acid comprises DNA encoding the PRO220 polypeptide having aminoacid residues 1 through 708 of FIG. 26 (SEQ ID NO:69), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. Also provided herein is an isolated nucleic acidcomprises DNA encoding the PRO221 polypeptide having amino acid residues1 through 259 of FIG. 28 (SEQ ID NO:71), or is complementary to suchencoding nucleic acid sequence, and remains stably bound to it under atleast moderate, and optionally, under high stringency conditions.Moreover, also provided herein is an isolated nucleic acid comprises DNAencoding the PRO227 polypeptide having amino acid residues 1 through 620of FIG. 30 (SEQ ID NO:73), or is complementary to such encoding nucleicacid sequence, and remains stably bound to it under at least moderate,and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PRO220, PRO221and PRO227 polypeptides. in particular, provided herein is the isolatednative sequence for the PRO220 polypeptide, which in one embodiment,includes an amino acid sequence comprising residues 1 to 708 of FIG. 26(SEQ ID NO:69). Additionally provided herein is the isolated nativesequence for the PRO221 polypeptide, which in one embodiment, includesan amino acid sequence comprising residues 1 to 259 of FIG. 28 (SEQ IDNO:71). Moreover, provided herein is the isolated native sequence forthe PRO227 polypeptide, which in one embodiment, includes an amino acidsequence comprising residues 1 to 620 of FIG. 30 (SEQ ID NO:73).

12. PRO258

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to CRTAM and poliovirus receptor precursors, wherein thepolypeptide is designated in the present application as “PRO258”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO258 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO258 polypeptidehaving amino acid residues 1 to 398 of FIG. 32 (SEQ ID NO:84), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO258polypeptide. In particular, the invention provides isolated nativesequence PRO258 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 398 of FIG. 32 (SEQ ID NO:84). Anadditional embodiment of the present invention is directed to anisolated extracellular domain of a PRO258 polypeptide.

13. PRO266

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO266”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO266 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO266 polypeptidehaving amino acid residues 1 to 696 of FIG. 34 (SEQ ID NO:91), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO266polypeptide. In particular, the invention provides isolated nativesequence PRO266 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 696 of FIG. 34 (SEQ ID NO:91).

14. PRO269

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as PRO269.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO269 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO269 polypeptidehaving amino acid residues 1 to 490 of FIG. 36 (SEQ ID NO:96), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO269polypeptide. In particular, the invention provides isolated nativesequence PRO269 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 490 of FIG. 36 (SEQ ID NO:96). Anadditional embodiment of the present invention is directed to anisolated extracellular domain of a PRO269 polypeptide.

15. PRO287

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO287”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO287 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO287 polypeptidehaving amino acid residues 1 to 415 of FIG. 38 (SEQ ID NO:104), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO287polypeptide. In particular, the invention provides isolated nativesequence PRO287 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 415 of FIG. 38 (SEQ ID NO:104).

16. PRO214

Applicants have identified a cDNA clone that encodes a novelpolypeptide, designated in the present application as “PRO214”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO214 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO214 polypeptideof FIG. 40 (SEQ ID NO:109), or is complementary to such encoding nucleicacid sequence, and remains stably bound to it under at least moderate,and optionally, under high stringency conditions. In another aspect, theinvention provides a nucleic acid comprising the coding sequence of FIG.39 (SEQ ID NO:108) or its complement. In another aspect, the inventionprovides a nucleic acid of the full length protein of cloneDNA32286-1191, deposited with ATCC under accession number ATCC 209385.

In yet another embodiment, the invention provides isolated PRO214polypeptide. In particular, the invention provides isolated nativesequence PRO214 polypeptide, which in one embodiment, includes an aminoacid sequence comprising the residues of FIG. 40 (SEQ ID NO:109).Alternatively, the invention provides a polypeptide encoded by thenucleic acid deposited under accession number ATCC 209385.

17. PRO317

Applicants have identified a cDNA clone that encodes a novelpolypeptide, designated in the present application as “PRO317”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding PRO317 polypeptide. In one aspect, theisolated nucleic acid comprises DNA (SEQ ID NO:113) encoding PRO317polypeptide having amino acid residues 1 to 366 of FIG. 42, or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO317polypeptide. In particular, the invention provides isolatednative-sequence PRO317 polypeptide, which in one embodiment, includes anamino acid sequence comprising residues 1 to 366 of FIG. 42 (SEQ IDNO:114).

In yet another embodiment, the invention supplies a method of detectingthe presence of PRO317 in a sample, the method comprising:

-   -   a) contacting a detectable anti-PRO317 antibody with a sample        suspected of containing PRO317; and    -   b) detecting binding of the antibody to the sample; wherein the        sample is selected from the group consisting of a body fluid, a        tissue sample, a cell extract, and a cell culture medium.

In a still further embodiment a method is provided for determining thepresence of PRO317 mRNA in a sample, the method comprising:

-   -   a) contacting a sample suspected of containing PRO317 mRNA with        a detectable nucleic acid probe that hybridizes under moderate        to stringent conditions to PRO317 mRNA; and    -   b) detecting hybridization of the probe to the sample.

Preferably, in this method the sample is a tissue sample and thedetecting step is by in situ hybridization, or the sample is a cellextract and detection is by Northern analysis.

Further, the invention provides a method for treating aPRO317-associated disorder comprising administering to a mammal aneffective amount of the PRO317 polypeptide or a composition thereofcontaining a carrier, or with an effective amount of a PRO317 agonist orPRO317 antagonist, such as an antibody which binds specifically toPRO317.

18. PRO301

Applicants have identified a cDNA clone (DNA40628-1216) that encodes anovel polypeptide, designated in the present application as “PRO301 ”.

In one embodiment, the invention provides an isolated nucleic acidmolecule having at least about 80% sequence identity to (a) a DNAmolecule encoding a PRO301 polypeptide comprising the sequence of aminoacids 28 to 258 of FIG. 44 (SEQ ID NO:119), or (b) the complement of theDNA molecule of (a). The sequence identity preferably is about 85%, morepreferably about 90%, most preferably about 95%. In one aspect, theisolated nucleic acid has at least about 80%, preferably at least about85%, more preferably at least about 90%, and most preferably at leastabout 95% sequence identity with a polypeptide having amino acidresidues 28 to 258 of FIG. 44 (SEQ ID NO:119). Preferably, the highestdegree of sequence identity occurs within the extracellular domains(amino acids 28 to 258 of FIG. 44, SEQ ID NO:119). In a furtherembodiment, the isolated nucleic acid molecule comprises DNA encoding aPRO301 polypeptide having amino acid residues 28 to 299 of FIG. 44 (SEQID NO:119), or is complementary to such encoding nucleic acid sequence,and remains stably bound to it under at least moderate, and optionally,under high stringency conditions. In another aspect, the inventionprovides a nucleic acid of the full length protein of cloneDNA40628-1216, deposited with the ATCC under accession number ATCC209432, alternatively the coding sequence of clone DNA40628-1216,deposited under accession number ATCC 209432.

In yet another embodiment, the invention provides isolated PRO301polypeptide. In particular, the invention provides isolated nativesequence PRO301 polypeptide, which in one embodiment, includes an aminoacid sequence comprising the extracellular domain residues 28 to 258 ofFIG. 44 (SEQ ID NO:119). Native PRO301 polypeptides with or without thenative signal sequence (amino acids 1 to 27 in FIG. 44 (SEQ ID NO:119),and with or without the initiating methionine are specifically included.Additionally, the sequences of the invention may also comprise thetransmembrane domain (residues 236 to about 258 in FIG. 44; SEQ IDNO:119) and/or the intracellular domain (about residue 259 to 299 inFIG. 44; SEQ ID NO:119). Alternatively, the invention provides a PRO301polypeptide encoded by the nucleic acid deposited under accession numberATCC 209432.

19. PRO224

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO224”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO224 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO224 polypeptidehaving amino acid residues 1 to 282 of FIG. 46 (SEQ ID NO:127), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO224polypeptide. In particular, the invention provides isolated nativesequence PRO224 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 282 of FIG. 46 (SEQ ID NO:127).

20. PRO222

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO222”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO222 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO222 polypeptidehaving amino acid residues 1 to 490 of FIG. 48 (SEQ ID NO:132), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO222polypeptide. In particular, the invention provides isolated nativesequence PRO222 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 490 of FIG. 48 (SEQ ID NO:132).

21. PRO234

Applicants have identified a cDNA clone that encodes a novel lectinpolypeptide molecule, designated in the present application as “PRO234”.

In one embodiment, the invention provides an isolated nucleic acidencoding a novel lectin comprising DNA encoding a PRO234 polypeptide. Inone aspect, the isolated nucleic acid comprises the DNA encoding PRO234polypeptides having amino acid residues 1 to 382 of FIG. 50 (SEQ IDNO:137), or is complementary to such encoding nucleic acid sequence, andremains stably bound to it under at least moderate, and optionally,under high stringency conditions. In another aspect, the inventionprovides an isolated nucleic acid molecule comprising the nucleotidesequence of FIG. 49 (SEQ ID NO:136).

In another embodiment, the invention provides isolated novel PRO234polypeptides. In particular, the invention provides isolated nativesequence PRO234 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 382 of FIG. 50 (SEQ ID NO:137).

In yet another embodiment, the invention provides oligonucleotide probesuseful for isolating genomic and cDNA nucleotide sequences.

22. PRO231

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to a putative acid phosphatase, wherein the polypeptideis designated in the present application as “PRO231”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO231 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO231 polypeptidehaving amino acid residues 1 to 428 of FIG. 52 (SEQ ID NO:142), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO231polypeptide. In particular, the invention provides isolated nativesequence PRO231 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 428 of FIG. 52 (SEQ ID NO:142).

23. PRO229

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to scavenger receptors wherein the polypeptide isdesignated in the present application as “PRO229”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO229 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO229 polypeptidehaving amino acid residues 1 to 347 of FIG. 54 (SEQ ID NO:148), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO229polypeptide. In particular, the invention provides isolated nativesequence PRO229 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 347 of FIG. 54 (SEQ ID NO:148).

24. PRO238

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to reductase, wherein the polypeptide is designated inthe present application as “PRO238”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO238 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO238 polypeptidehaving amino acid residues 1 to 310 of FIG. 56 (SEQ ID NO:153), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO238polypeptide. In particular, the invention provides isolated nativesequence PRO238 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 310 of FIG. 56 (SEQ ID NO:153).

25. PRO233

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO233”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO233 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO233 polypeptidehaving amino acid residues 1 to 300 of FIG. 58 (SEQ ID NO:159), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO233polypeptide. In particular, the invention provides isolated nativesequence PRO233 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 300 of FIG. 58 (SEQ ID NO:159).

26. PRO223

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to serine carboxypeptidase polypeptides, wherein thepolypeptide is designated in the present application as “PRO223 ”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO223 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO223 polypeptidehaving amino acid residues 1 to 476 of FIG. 60 (SEQ ID NO:164), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO223polypeptide. In particular, the invention provides isolated nativesequence PRO223 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 476 of FIG. 60 (SEQ ID NO:164).

27. PRO235

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO235”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO235 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO235 polypeptidehaving amino acid residues 1 to 552 of FIG. 62 (SEQ ID NO:170), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO235polypeptide. In particular, the invention provides isolated nativesequence PRO235 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 552 of FIG. 62 (SEQ ID NO:170).

28. PRO236 and PRO262

Applicants have identified cDNA clones that encode novel polypeptideshaving homology to β-galactosidase, wherein those polypeptides aredesignated in the present application as “PRO236” and “PRO262”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO236 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO236 polypeptidehaving amino acid residues 1 to 636 of FIG. 64 (SEQ ID NO:175), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO262 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO262 polypeptidehaving amino acid residues 1 to 654 of FIG. 66 (SEQ ID NO:177), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO236polypeptide. In particular, the invention provides isolated nativesequence PRO236 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 636 of FIG. 64 (SEQ ID NO:175).

In another embodiment, the invention provides isolated PRO262polypeptide. In particular, the invention provides isolated nativesequence PRO262 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 654 of FIG. 66 (SEQ ID NO:177).

29. PRO239

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO239”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO239 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO239 polypeptidehaving amino acid residues 1 to 501 of FIG. 68 (SEQ ID NO:185), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO239polypeptide. In particular, the invention provides isolated nativesequence PRO239 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 501 of FIG. 68 (SEQ ID NO:185).

30. PRO257

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO257”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO257 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO257 polypeptidehaving amino acid residues 1 to 607 of FIG. 70 (SEQ ID NO:190), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO257polypeptide. In particular, the invention provides isolated nativesequence PRO257 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 607 of FIG. 70 (SEQ ID NO:190).An additional embodiment of the present invention is directed to anisolated extracellular domain of a PRO257 polypeptide.

31. PRO260

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO260”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO260 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO260 polypeptidehaving amino acid residues 1 to 467 of FIG. 72 (SEQ ID NO:195), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO260polypeptide. In particular, the invention provides isolated nativesequence PRO260 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 467 of FIG. 72 (SEQ ID NO:195).

32. PRO263

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to CD44 antigen, wherein the polypeptide is designatedin the present application as “PRO263”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO263 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO263 polypeptidehaving amino acid residues 1 to 322 of FIG. 74 (SEQ ID NO:201), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO263polypeptide. In particular, the invention provides isolated nativesequence PRO263 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 322 of FIG. 74 (SEQ ID NO:201).An additional embodiment of the present invention is directed to anisolated extracellular domain of a PRO263 polypeptide.

33. PRO270

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO270”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO270 polypeptide. In one aspect,the isolated nucleic acid comprises DNA which includes the sequenceencoding the PRO270 polypeptide having amino acid residues 1 to 296 ofFIG. 76 (SEQ ID NO:207), or is complementary to such encoding nucleicacid sequence, and remains stably bound to it under at least moderate,and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PRO270polypeptide. In particular, the invention provides isolated nativesequence PRO270 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 296 of FIG. 76 (SEQ ID NO:207).

34. PRO271

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to the proteoglycan link protein, wherein thepolypeptide is designated in the present application as “PRO271”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO271 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO271 polypeptidehaving amino acid residues 1 to 360 of FIG. 78 (SEQ ID NO:213), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO271polypeptide. In particular, the invention provides isolated nativesequence PRO271 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 360 of FIG. 78 (SEQ ID NO:213).

35. PRO272

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO272”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO272 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO272 polypeptidehaving amino acid residues 1 to 328 of FIG. 80 (SEQ ID NO:221), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO272polypeptide. In particular, the invention provides isolated nativesequence PRO272 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 328 of FIG. 80 (SEQ ID NO:211).

36. PRO294

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO294”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO294 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO294 polypeptidehaving amino acid residues 1 to 550 of FIG. 82 (SEQ ID NO:227), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO294polypeptide. In particular, the invention provides isolated nativesequence PRO294 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 550 of FIG. 82 (SEQ ID NO:227).

37. PRO295

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO295”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO295 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO295 polypeptidehaving amino acid residues 1 to 350 of FIG. 84 (SEQ ID NO:236), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO295polypeptide. In particular, the invention provides isolated nativesequence PRO295 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 350 of FIG. 84 (SEQ ID NO:236).

38. PRO293

Applicants have identified a cDNA clone that encodes a novel humanneuronal leucine rich repeat polypeptide, wherein the polypeptide isdesignated in the present application as “PRO293”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO293 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO293 polypeptidehaving amino acid residues 1 to 713 of FIG. 86 (SEQ ID NO:245), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO293polypeptide. In particular, the invention provides isolated nativesequence PRO293 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 713 of FIG. 86 (SEQ ID NO:245).An additional embodiment of the present invention is directed to anisolated extracellular domain of a PRO293 polypeptide.

39. PRO247

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving leucine rich repeats wherein the polypeptide is designated in thepresent application as “PRO247”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO247 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO247 polypeptidehaving amino acid residues 1 to 546 of FIG. 88 (SEQ ID NO:250), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO247polypeptide. In particular, the invention provides isolated nativesequence PRO247 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 546 of FIG. 88 (SEQ ID NO:250).An additional embodiment of the present invention is directed to anisolated extracellular domain of a PRO247 polypeptide.

40. PRO302, PRO303, PRO304, PRO307 and PRO343

Applicants have identified cDNA clones that encode novel polypeptideshaving homology to various proteases, wherein those polypeptide aredesignated in the present application as “PRO302”, “PRO303”, “PRO304”,“PRO307” and “PRO343” polypeptides.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO302 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO302 polypeptidehaving amino acid residues 1 to 452 of FIG. 90 (SEQ ID NO:255), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO303 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO303 polypeptidehaving amino acid residues 1 to 314 of FIG. 92 (SEQ ID NO:257), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In yet another embodiment, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO304 polypeptide. In oneaspect, the isolated nucleic acid comprises DNA encoding the PRO304polypeptide having amino acid residues 1 to 556 of FIG. 94 (SEQ IDNO:259), or is complementary to such encoding nucleic acid sequence, andremains stably bound to it under at least moderate, and optionally,under high stringency conditions.

In another embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO307 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO307 polypeptidehaving amino acid residues 1 to 383 of FIG. 96 (SEQ ID NO:261), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO343 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO343 polypeptidehaving amino acid residues 1 to 317 of FIG. 98 (SEQ ID NO:263), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO302polypeptide. In particular, the invention provides isolated nativesequence PRO302 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 452 of FIG. 90 (SEQ ID NO:255).In another embodiment, the invention provides isolated PRO303polypeptide. In particular, the invention provides isolated nativesequence PRO303 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 314 of FIG. 92 (SEQ ID NO:257).

In another embodiment, the invention provides isolated PRO304polypeptide. In particular, the invention provides isolated nativesequence PRO304 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 556 of FIG. 94 (SEQ ID NO:259).

In another embodiment, the invention provides isolated PRO307polypeptide. In particular, the invention provides isolated nativesequence PRO307 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 383 of FIG. 96 (SEQ ID NO:261).

In another embodiment, the invention provides isolated PRO343polypeptide. In particular, the invention provides isolated nativesequence PRO343 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 317 of FIG. 98 (SEQ ID NO:263).

41. PRO328

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO328”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO328 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO328 polypeptidehaving amino acid residues 1 to 463 of FIG. 100 (SEQ ID NO:285), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO328polypeptide. In particular, the invention provides isolated nativesequence PRO328 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 463 of FIG. 100 (SEQ ID NO:285).An additional embodiment of the present invention is directed to anisolated extracellular domain of a PRO306 polypeptide.

42. PRO335, PRO331 and PRO326

Applicants have identified three cDNA clones that respectively encodethree novel polypeptides, each having leucine rich repeats and homologyto LIG-1 and ALS. These polypeptides are designated in the presentapplication as PRO335, PRO331 and PRO326, respectively.

In one embodiment, the invention provides three isolated nucleic acidmolecules comprising DNA respectively encoding PRO335, PRO331 andPRO326, respectively. In one aspect, herein is provided an isolatednucleic acid comprising DNA encoding the PRO335 polypeptide having aminoacid residues 1 through 1059 of FIG. 102 (SEQ ID NO:290), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. Also provided herein is an isolated nucleic acidcomprises DNA encoding the PRO331 polypeptide having amino acid residues1 through 640 of FIG. 104 (SEQ ID NO:292), or is complementary to suchencoding nucleic acid sequence, and remains stably bound to it under atleast moderate, and optionally, under high stringency conditions.Additionally provided herein is an isolated nucleic acid comprises DNAencoding the PRO326 polypeptide having amino acid residues 1 through1119 of FIG. 106 (SEQ ID NO:294), or is complementary to such encodingnucleic acid sequence, and remains stably bound to it under at leastmoderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PRO335, PRO331and PRO326 polypeptides or extracellular domains thereof. In particular,the invention provides isolated native sequence for the PRO335polypeptide, which in one embodiment, includes an amino acid sequencecomprising residues 1 through 1059 of FIG. 102 (SEQ ID NO:290). Alsoprovided herein is the isolated native sequence for the PRO331polypeptide, which in one embodiment, includes an amino acid sequencecomprising residues 1 through 640 of FIG. 104 (SEQ ID NO:292). Alsoprovided herein is the isolated native sequence for the PRO326polypeptide, which in one embodiment, includes an amino acid sequencecomprising residues 1 through 1119 of FIG. 106 (SEQ ID NO:294).

43. PRO332

Applicants have identified a cDNA clone (DNA40982-1235) that encodes anovel polypeptide, designated in the present application as “PRO332.”

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA having at least about 80% sequence identity to(a) a DNA molecule encoding a PRO358 polypeptide comprising the sequenceof amino acids 49 to 642 of FIG. 108 (SEQ ID NO:310), or (b) thecomplement of the DNA molecule of (a). The sequence identity preferablyis about 85%, more preferably about 90%, most preferably about 95%. Inone aspect, the isolated nucleic acid has at least about 80%, preferablyat least about 85%, more preferably at least about 90%, and mostpreferably at least about 95% sequence identity with a polypeptidehaving amino acid residues 1 to 642 of FIG. 108 (SEQ ID NO:310).Preferably, the highest degree of sequence identity occurs within theleucine-rich repeat domains (amino acids 116 to 624 of FIG. 108, SEQ IDNO:310). In a further embodiment, the isolated nucleic acid moleculecomprises DNA encoding a PRO332 polypeptide having amino acid residues49 to 642 of FIG. 108 (SEQ ID NO:310), or is complementary to suchencoding nucleic acid sequence, and remains stably bound to it under atleast moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PRO332polypeptides. In particular, the invention provides isolated nativesequence PRO332 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 49 to 624 of FIG. 108 (SEQ ID NO:310).Native PRO332 polypeptides with or without the native signal sequence(amino acids 1 to 48 in FIG. 108, SEQ ID NO:310), and with or withoutthe initiating methionine are specifically included.

44. PRO334

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to fibulin and fibrillin, wherein the polypeptide isdesignated in the present application as “PRO334”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO334 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO334 polypeptidehaving amino acid residues 1 to 509 of FIG. 110 (SEQ ID NO:315), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO334polypeptide. In particular, the invention provides isolated nativesequence PRO334 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 509 of FIG. 110 (SEQ ID NO:315).

45. PRO346

Applicants have identified a cDNA clone (DNA44167-1243) that encodes anovel polypeptide, designated in the present application as “PRO346.” Inone embodiment, the invention provides an isolated nucleic acid moleculehaving at least about 80% sequence identity to (a) a DNA moleculeencoding a PRO346 polypeptide comprising the sequence of amino acids 19to 339 of FIG. 112 (SEQ ID NO:320), or (b) the complement of the DNAmolecule of (a). The sequence identity preferably is about 85%, morepreferably about 90%, most preferably about 95%. In one aspect, theisolated nucleic acid has at least about 80%, preferably at least about85%, more preferably at least about 90%, and most preferably at leastabout 95% sequence identity with a polypeptide having amino acidresidues 19 to 339 of FIG. 112 (SEQ ID NO:320). Preferably, the highestdegree of sequence identity occurs within the extracellular domains(amino acids 19 to 339 of FIG. 112, SEQ ID NO:320). In alternativeembodiments, the polypeptide by which the homology is measured comprisesthe residues 1-339, 19-360 or 19-450 of FIG. 112, SEQ ID NO:320). In afurther embodiment, the isolated nucleic acid molecule comprises DNAencoding a PRO346 polypeptide having amino acid residues 19 to 339 ofFIG. 112 (SEQ ID NO:320), alternatively residues 1-339, 19-360 or 19-450of FIG. 112 (SEQ ID NO:320) or is complementary to such encoding nucleicacid sequence, and remains stably bound to it under at least moderate,and optionally, under high stringency conditions. In another aspect, theinvention provides a nucleic acid of the full length protein of cloneDNA44167-1243, deposited with the ATCC under accession number ATCC209434, alternatively the coding sequence of clone DNA44167-1243,deposited under accession number ATCC 209434.

In yet another embodiment, the invention provides isolated PRO346polypeptide. In particular, the invention provides isolated nativesequence PRO346 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 19 to 339 of FIG. 112 (SEQ ID NO:320).Native PRO346 polypeptides with or without the native signal sequence(residues 1 to 18 in FIG. 112 (SEQ ID NO:320), with or without theinitiating methionine, with or without the transmembrane domain(residues 340 to 360) and with or without the intracellular domain(residues 361 to 450) are specifically included. Alternatively, theinvention provides a PRO346 polypeptide encoded by the nucleic aciddeposited under accession number ATCC 209434.

46. PRO268

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to protein disulfide isomerase, wherein the polypeptideis designated in the present application as “PRO268”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO268 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO268 polypeptidehaving amino acid residues 1 to 280 of FIG. 114 (SEQ ID NO:325), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO268polypeptide. In particular, the invention provides isolated nativesequence PRO268 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 280 of FIG. 114 (SEQ ID NO:325).An additional embodiment of the present invention is directed to anisolated extracellular domain of a PRO268 polypeptide.

47. PRO330

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to the alpha subunit of prolyl 4-hydroxylase, whereinthe polypeptide is designated in the present application as “PRO330”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO330 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO330 polypeptidehaving amino acid residues 1 to 533 of FIG. 116 (SEQ ID NO:332), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO330polypeptide. In particular, the invention provides isolated nativesequence PRO330 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 533 of FIG. 116 (SEQ ID NO:332).

48. PRO339 and PRO310

Applicants have identified two cDNA clones wherein each clone encodes anovel polypeptide having homology to fringe, wherein the polypeptidesare designated in the present application as “PRO339” and “PRO310”.

In one embodiment, the invention provides isolated nucleic acidmolecules comprising DNA encoding a PRO339 and/or a PRO310 polypeptide.In one aspect, the isolated nucleic acid comprises DNA encoding thePRO339 polypeptide having amino acid residues 1 to 772 of FIG. 118 (SEQID NO:339), or is complementary to such encoding nucleic acid sequence,and remains stably bound to it under at least moderate, and optionally,under high stringency conditions. In another aspect, the isolatednucleic acid comprises DNA encoding the PRO310 polypeptide having aminoacid residues 1 to 318 of FIG. 120 (SEQ ID NO:341), or is complementaryto such encoding nucleic acid sequence, and remains stably bound to itunder at least moderate, and optionally, under high stringencyconditions.

In another embodiment, the invention provides isolated PRO339 as well asisolated PRO310 polypeptides. In particular, the invention providesisolated native sequence PRO339 polypeptide, which in one embodiment,includes an amino acid sequence comprising residues 1 to 772 of FIG. 118(SEQ ID NO:339). The invention further provides isolated native sequencePRO310 polypeptide, which in one embodiment, includes an amino acidsequence comprising residues 1 to 318 of FIG. 120 (SEQ ID NO:341).

49. PRO244

Applicants have identified a cDNA clone that encodes a novelpolypeptide, designated in the present application as “PRO244”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding PRO244 polypeptide. In one aspect, theisolated nucleic acid comprises DNA encoding PRO244 polypeptide havingamino acid residues 1 to 219 of FIG. 122 (SEQ ID NO:377), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO244polypeptide. In particular, the invention provides isolated nativesequence PRO244 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 219 of FIG. 122 (SEQ ID NO:377).

50. Additional Embodiments

In other embodiments of the present invention, the invention providesvectors comprising DNA encoding any of the herein describedpolypeptides. Host cell comprising any such vector are also provided. Byway of example, the host cells may be CHO cells, E. coli, or yeast. Aprocess for producing any of the herein described polypeptides isfurther provided and comprises culturing host cells under conditionssuitable for expression of the desired polypeptide and recovering thedesired polypeptide from the cell culture.

In other embodiments, the invention provides chimeric moleculescomprising any of the herein described polypeptides fused to aheterologous polypeptide or amino acid sequence. Example of suchchimeric molecules comprise any of the herein described polypeptidesfused to an epitope tag sequence or a Fc region of an immunoglobulin.

In another embodiment, the invention provides an antibody whichspecifically binds to any of the above or below described polypeptides.Optionally, the antibody is a monoclonal antibody, humanized antibody,antibody fragment or single-chain antibody.

In yet other embodiments, the invention provides oligonucleotide probesuseful for isolating genomic and cDNA nucleotide sequences, whereinthose probes may be derived from any of the above or below describednucleotide sequences.

In other embodiments, the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence that encodes a PROpolypeptide.

In one aspect, the isolated nucleic acid molecule comprises a nucleotidesequence having at least about 80% sequence identity, preferably atleast about 81% sequence identity, more preferably at least about 82%sequence identity, yet more preferably at least about 83% sequenceidentity, yet more preferably at least about 84% sequence identity, yetmore preferably at least about 85% sequence identity, yet morepreferably at least about 86% sequence identity, yet more preferably atleast about 87% sequence identity, yet more preferably at least about88% sequence identity, yet more preferably at least about 89% sequenceidentity, yet more preferably at least about 90% sequence identity, yetmore preferably at least about 91% sequence identity, yet morepreferably at least about 92% sequence identity, yet more preferably atleast about 93% sequence identity, yet more preferably at least about94% sequence identity, yet more preferably at least about 95% sequenceidentity, yet more preferably at least about 96% sequence identity, yetmore preferably at least about 97% sequence identity, yet morepreferably at least about 98% sequence identity and yet more preferablyat least about 99% sequence identity to (a) a DNA molecule encoding aPRO polypeptide having a full-length amino acid sequence as disclosedherein, an amino acid sequence lacking the signal peptide as disclosedherein or an extracellular domain of a transmembrane protein, with orwithout the signal peptide, as disclosed herein, or (b) the complementof the DNA molecule of (a).

In other aspects, the isolated nucleic acid molecule comprises anucleotide sequence having at least about 80% sequence identity,preferably at least about 81% sequence identity, more preferably atleast about 82% sequence identity, yet more preferably at least about83% sequence identity, yet more preferably at least about 84% sequenceidentity, yet more preferably at least about 85% sequence identity, yetmore preferably at least about 86% sequence identity, yet morepreferably at least about 87% sequence identity, yet more preferably atleast about 88% sequence identity, yet more preferably at least about89% sequence identity, yet more preferably at least about 90% sequenceidentity, yet more preferably at least about 91% sequence identity, yetmore preferably at least about 92% sequence identity, yet morepreferably at least about 93% sequence identity, yet more preferably atleast about 94% sequence identity, yet more preferably at least about95% sequence identity, yet more preferably at least about 96% sequenceidentity, yet more preferably at least about 97% sequence identity, yetmore preferably at least about 98% sequence identity and yet morepreferably at least about 99% sequence identity to (a) a DNA moleculecomprising the coding sequence of a full-length PRO polypeptide cDNA asdisclosed herein, the coding sequence of a PRO polypeptide lacking thesignal peptide as disclosed herein or the coding sequence of anextracellular domain of a transmembrane PRO polypeptide, with or withoutthe signal peptide, as disclosed herein, or (b) the complement of theDNA molecule of (a).

In a further aspect, the invention concerns an isolated nucleic acidmolecule comprising a nucleotide sequence having at least about 80%sequence identity, preferably at least about 81% sequence identity, morepreferably at least about 82% sequence identity, yet more preferably atleast about 83% sequence identity, yet more preferably at least about84% sequence identity, yet more preferably at least about 85% sequenceidentity, yet more preferably at least about 86% sequence identity, yetmore preferably at least about 87% sequence identity, yet morepreferably at least about 88% sequence identity, yet more preferably atleast about 89% sequence identity, yet more preferably at least about90% sequence identity, yet more preferably at least about 91% sequenceidentity, yet more preferably at least about 92% sequence identity, yetmore preferably at least about 93% sequence identity, yet morepreferably at least about 94% sequence identity, yet more preferably atleast about 95% sequence identity, yet more preferably at least about96% sequence identity, yet more preferably at least about 97% sequenceidentity, yet more preferably at least about 98% sequence identity andyet more preferably at least about 99% sequence identity to (a) a DNAmolecule that encodes the same mature polypeptide encoded by any of thehuman protein cDNAs deposited with the ATCC as disclosed herein, or (b)the complement of the DNA molecule of (a).

Another aspect the invention provides an isolated nucleic acid moleculecomprising a nucleotide sequence encoding a PRO polypeptide which iseither transmembrane domain-deleted or transmembrane domain-inactivated,or is complementary to such encoding nucleotide sequence, wherein thetransmembrane domain(s) of such polypeptide are disclosed herein.Therefore, soluble extracellular domains of the herein described PROpolypeptides are contemplated.

Another embodiment is directed to fragments of a PRO polypeptide codingsequence, or the complement thereof, that may find use as, for example,hybridization probes or for encoding fragments of a PRO polypeptide thatmay optionally encode a polypeptide comprising a binding site for ananti-PRO antibody. Such nucleic acid fragments are usually at leastabout 20 nucleotides in length, preferably at least about 30 nucleotidesin length, more preferably at least about 40 nucleotides in length, yetmore preferably at least about 50 nucleotides in length, yet morepreferably at least about 60 nucleotides in length, yet more preferablyat least about 70 nucleotides in length, yet more preferably at leastabout 80 nucleotides in length, yet more preferably at least about 90nucleotides in length, yet more preferably at least about 100nucleotides in length, yet more preferably at least about 110nucleotides in length, yet more preferably at least about 120nucleotides in length, yet more preferably at least about 130nucleotides in length, yet more preferably at least about 140nucleotides in length, yet more preferably at least about 150nucleotides in length, yet more preferably at least about 160nucleotides in length, yet more preferably at least about 170nucleotides in length, yet more preferably at least about 180nucleotides in length, yet more preferably at least about 190nucleotides in length, yet more preferably at least about 200nucleotides in length, yet more preferably at least about 250nucleotides in length, yet more preferably at least about 300nucleotides in length, yet more preferably at least about 350nucleotides in length, yet more preferably at least about 400nucleotides in length, yet more preferably at least about 450nucleotides in length, yet more preferably at least about 500nucleotides in length, yet more preferably at least about 600nucleotides in length, yet more preferably at least about 700nucleotides in length, yet more preferably at least about 800nucleotides in length, yet more preferably at least about 900nucleotides in length and yet more preferably at least about 1000nucleotides in length, wherein in this context the term “about” meansthe referenced nucleotide sequence length plus or minus 10% of thatreferenced length. It is noted that novel fragments of a PROpolypeptide-encoding nucleotide sequence may be determined in a routinemanner by aligning the PRO polypeptide-encoding nucleotide sequence withother known nucleotide sequences using any of a number of well knownsequence alignment programs and determining which PROpolypeptide-encoding nucleotide sequence fragment(s) are novel. All ofsuch PRO polypeptide-encoding nucleotide sequences are contemplatedherein. Also contemplated are the PRO polypeptide fragments encoded bythese nucleotide molecule fragments, preferably those PRO polypeptidefragments that comprise a binding site for an anti-PRO antibody.

In another embodiment, the invention provides isolated PRO polypeptideencoded by any of the isolated nucleic acid sequences hereinaboveidentified.

In a certain aspect, the invention concerns an isolated PRO polypeptide,comprising an amino acid sequence having at least about 80% sequenceidentity, preferably at least about 81% sequence identity, morepreferably at least about 82% sequence identity, yet more preferably atleast about 83% sequence identity, yet more preferably at least about84% sequence identity, yet more preferably at least about 85% sequenceidentity, yet more preferably at least about 86% sequence identity, yetmore preferably at least about 87% sequence identity, yet morepreferably at least about 88% sequence identity, yet more preferably atleast about 89% sequence identity, yet more preferably at least about90% sequence identity, yet more preferably at least about 91% sequenceidentity, yet more preferably at least about 92% sequence identity, yetmore preferably at least about 93% sequence identity, yet morepreferably at least about 94% sequence identity, yet more preferably atleast about 95% sequence identity, yet more preferably at least about96% sequence identity, yet more preferably at least about 97% sequenceidentity, yet more preferably at least about 98% sequence identity andyet more preferably at least about 99% sequence identity to a PROpolypeptide having a full-length amino acid sequence as disclosedherein, an amino acid sequence lacking the signal peptide as disclosedherein or an extracellular domain of a transmembrane protein, with orwithout the signal peptide, as disclosed herein.

In a further aspect, the invention concerns an isolated PRO polypeptidecomprising an amino acid sequence having at least about 80% sequenceidentity, preferably at least about 81% sequence identity, morepreferably at least about 82% sequence identity, yet more preferably atleast about 83% sequence identity, yet more preferably at least about84% sequence identity, yet more preferably at least about 85% sequenceidentity, yet more preferably at least about 86% sequence identity, yetmore preferably at least about 87% sequence identity, yet morepreferably at least about 88% sequence identity, yet more preferably atleast about 89% sequence identity, yet more preferably at least about90% sequence identity, yet more preferably at least about 91% sequenceidentity, yet more preferably at least about 92% sequence identity, yetmore preferably at least about 93% sequence identity, yet morepreferably at least about 94% sequence identity, yet more preferably atleast about 95% sequence identity, yet more preferably at least about96% sequence identity, yet more preferably at least about 97% sequenceidentity, yet more preferably at least about 98% sequence identity andyet more preferably at least about 99% sequence identity to an aminoacid sequence encoded by any of the human protein cDNAs deposited withthe ATCC as disclosed herein.

In a further aspect, the invention concerns an isolated PRO polypeptidecomprising an amino acid sequence scoring at least about 80% positives,preferably at least about 81% positives, more preferably at least about82% positives, yet more preferably at least about 83% positives, yetmore preferably at least about 84% positives, yet more preferably atleast about 85% positives, yet more preferably at least about 86%positives, yet more preferably at least about 87% positives, yet morepreferably at least about 88% positives, yet more preferably at leastabout 89% positives, yet more preferably at least about 90% positives,yet more preferably at least about 91% positives, yet more preferably atleast about 92% positives, yet more preferably at least about 93%positives, yet more preferably at least about 94% positives, yet morepreferably at least about 95% positives, yet more preferably at leastabout 96% positives, yet more preferably at least about 97% positives,yet more preferably at least about 98% positives and yet more preferablyat least about 99% positives when compared with the amino acid sequenceof a PRO polypeptide having a full-length amino acid sequence asdisclosed herein, an amino acid sequence lacking the signal peptide asdisclosed herein or an extracellular domain of a transmembrane protein,with or without the signal peptide, as disclosed herein.

In a specific aspect, the invention provides an isolated PRO polypeptidewithout the N-terminal signal sequence and/or the initiating methionineand is encoded by a nucleotide sequence that encodes such an amino acidsequence as hereinbefore described. Processes for producing the same arealso herein described, wherein those processes comprise culturing a hostcell comprising a vector which comprises the appropriate encodingnucleic acid molecule under conditions suitable for expression of thePRO polypeptide and recovering the PRO polypeptide from the cellculture.

Another aspect the invention provides an isolated PRO polypeptide whichis either transmembrane domain-deleted or transmembranedomain-inactivated. Processes for producing the same are also hereindescribed, wherein those processes comprise culturing a host cellcomprising a vector which comprises the appropriate encoding nucleicacid molecule under conditions suitable for expression of the PROpolypeptide and recovering the PRO polypeptide from the cell culture.

In yet another embodiment, the invention concerns agonists andantagonists of a native PRO polypeptide as defined herein. In aparticular embodiment, the agonist or antagonist is an anti-PRO antibodyor a small molecule.

In a further embodiment, the invention concerns a method of identifyingagonists or antagonists to a PRO polypeptide which comprise contactingthe PRO polypeptide with a candidate molecule and monitoring abiological activity mediated by said PRO polypeptide. Preferably, thePRO polypeptide is a native PRO polypeptide.

In a still further embodiment, the invention concerns a composition ofmatter comprising a PRO polypeptide, or an agonist or antagonist of aPRO polypeptide as herein described, or an anti-PRO antibody, incombination with a carrier. Optionally, the carrier is apharmaceutically acceptable carrier.

Another embodiment of the present invention is directed to the use of aPRO polypeptide, or an agonist or antagonist thereof as hereinbeforedescribed, or an anti-PRO antibody, for the preparation of a medicamentuseful in the treatment of a condition which is responsive to the PROpolypeptide, an agonist or antagonist thereof or an anti-PRO antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) of a native sequencePRO211 cDNA, wherein SEQ ID NO:1 is a clone designated herein as“DNA32292-1131”.

FIG. 2 shows the amino acid sequence (SEQ ID NO:2) derived from thecoding sequence of SEQ ID NO:1 shown in FIG. 1.

FIG. 3 shows a nucleotide sequence (SEQ ID NO:3) of a native sequencePRO217 cDNA, wherein SEQ ID NO:3 is a clone designated herein as“DNA33094-1131”.

FIG. 4 shows the amino acid sequence (SEQ ID NO:4) derived from thecoding sequence of SEQ ID NO:3 shown in FIG. 3.

FIG. 5 shows a nucleotide sequence (SEQ ID NO:11) of a native sequencePRO230 cDNA, wherein SEQ ID NO:11 is a clone designated herein as“DNA33223-1136”.

FIG. 6 shows the amino acid sequence (SEQ ID NO:12) derived from thecoding sequence of SEQ ID NO:11 shown in FIG. 5.

FIG. 7 shows a nucleotide sequence designated herein as DNA20088 (SEQ IDNO:13).

FIG. 8 shows a nucleotide sequence (SEQ ID NO:17) of a native sequencePRO232 cDNA, wherein SEQ ID NO:17 is a clone designated herein as“DNA34435-1140”.

FIG. 9 shows the amino acid sequence (SEQ ID NO:18) derived from thecoding sequence of SEQ ID NO:17 shown in FIG. 8.

FIG. 10 shows a nucleotide sequence (SEQ ID NO:22) of a native sequencePRO187 cDNA, wherein SEQ ID NO:22 is a clone designated herein as“DNA27864-1155”.

FIG. 11 shows the amino acid sequence (SEQ ID NO:23) derived from thecoding sequence of SEQ ID NO:22 shown in FIG. 10.

FIG. 12 shows a nucleotide sequence (SEQ ID NO:27) of a native sequencePRO265 cDNA, wherein SEQ ID NO:27 is a clone designated herein as“DNA36350-1158”.

FIG. 13 shows the amino acid sequence (SEQ ID NO:28) derived from thecoding sequence of SEQ ID NO:27 shown in FIG. 12.

FIG. 14 shows a nucleotide sequence (SEQ ID NO:33) of a native sequencePRO219 cDNA, wherein SEQ ID NO:33 is a clone designated herein as“DNA32290-1164”.

FIG. 15 shows the amino acid sequence (SEQ ID NO:34) derived from thecoding sequence of SEQ ID NO:33 shown in FIG. 14.

FIG. 16 shows a nucleotide sequence (SEQ ID NO:38) of a native sequencePRO246 cDNA, wherein SEQ ID NO:38 is a clone designated herein as“DNA35639-1172”.

FIG. 17 shows the amino acid sequence (SEQ ID NO:39) derived from thecoding sequence of SEQ ID NO:38 shown in FIG. 16.

FIG. 18 shows a nucleotide sequence (SEQ ID NO:48) of a native sequencePRO228 cDNA, wherein SEQ ID NO:48 is a clone designated herein as“DNA33092-1202”.

FIG. 19 shows the amino acid sequence (SEQ ID NO:49) derived from thecoding sequence of SEQ ID NO:48 shown in FIG. 18.

FIG. 20 shows a nucleotide sequence designated herein as DNA21951 (SEQID NO:50).

FIG. 21 shows a nucleotide sequence (SEQ ID NO:58) of a native sequencePRO533 cDNA, wherein SEQ ID NO:58 is a clone designated herein as“DNA49435-1219”.

FIG. 22 shows the amino acid sequence (SEQ ID NO:59) derived from thecoding sequence of SEQ ID NO:58 shown in FIG. 21.

FIG. 23 shows a nucleotide sequence (SEQ ID NO:63) of a native sequencePRO245 cDNA, wherein SEQ ID NO:63 is a clone designated herein as“DNA35638-1141”.

FIG. 24 shows the amino acid sequence (SEQ ID NO:64) derived from thecoding sequence of SEQ ID NO:63 shown in FIG. 23.

FIG. 25 shows a nucleotide sequence (SEQ ID NO:68) of a native sequencePRO220 cDNA, wherein SEQ ID NO:68 is a clone designated herein as“DNA32298-1132”.

FIG. 26 shows the amino acid sequence (SEQ ID NO:69) derived from thecoding sequence of SEQ ID NO:68 shown in FIG. 25.

FIG. 27 shows a nucleotide sequence (SEQ ID NO:70) of a native sequencePRO221 cDNA, wherein SEQ ID NO:70 is a clone designated herein as“DNA33089-1132”.

FIG. 28 shows the amino acid sequence (SEQ ID NO:71) derived from thecoding sequence of SEQ ID NO:70 shown in FIG. 27.

FIG. 29 shows a nucleotide sequence (SEQ ID NO:72) of a native sequencePRO227 cDNA, wherein SEQ ID NO:72 is a clone designated herein as“DNA33786-1132”.

FIG. 30 shows the amino acid sequence (SEQ ID NO:73) derived from thecoding sequence of SEQ ID NO:72 shown in FIG. 29.

FIG. 31 shows a nucleotide sequence (SEQ ID NO:83) of a native sequencePRO258 cDNA, wherein SEQ ID NO:83 is a clone designated herein as“DNA35918-1174”.

FIG. 32 shows the amino acid sequence (SEQ ID NO:84) derived from thecoding sequence of SEQ ID NO:83 shown in FIG. 31.

FIG. 33 shows a nucleotide sequence (SEQ ID NO:90) of a native sequencePRO266 cDNA, wherein SEQ ID NO:90 is a clone designated herein as“DNA37150-1178”.

FIG. 34 shows the amino acid sequence (SEQ ID NO:91) derived from thecoding sequence of SEQ ID NO:90 shown in FIG. 33.

FIG. 35 shows a nucleotide sequence (SEQ ID NO:95) of a native sequencePRO269 cDNA, wherein SEQ ID NO:95 is a clone designated herein as“DNA38260-1180”.

FIG. 36 shows the amino acid sequence (SEQ ID NO:96) derived from thecoding sequence of SEQ ID NO:95 shown in FIG. 35.

FIG. 37 shows a nucleotide sequence (SEQ ID NO:103) of a native sequencePRO287 cDNA, wherein SEQ ID NO:103 is a clone designated herein as“DNA39969-1185”.

FIG. 38 shows the amino acid sequence (SEQ ID NO:104) derived from thecoding sequence of SEQ ID NO:103 shown in FIG. 37.

FIG. 39 shows a nucleotide sequence (SEQ ID NO:108) of a native sequencePRO214 cDNA, wherein SEQ ID NO:108 is a clone designated herein as“DNA32286-1191”.

FIG. 40 shows the amino acid sequence (SEQ ID NO:109) derived from thecoding sequence of SEQ ID NO:108 shown in FIG. 39.

FIG. 41 shows a nucleotide sequence (SEQ ID NO:113) of a native sequencePRO317 cDNA, wherein SEQ ID NO:113 is a clone designated herein as“DNA33461-1199”.

FIG. 42 shows the amino acid sequence (SEQ ID NO:114) derived from thecoding sequence of SEQ ID NO:113 shown in FIG. 41.

FIG. 43 shows a nucleotide sequence (SEQ ID NO:118) of a native sequencePRO301 cDNA, wherein SEQ ID NO:118 is a clone designated herein as“DNA40628-1216”.

FIG. 44 shows the amino acid sequence (SEQ ID NO:119) derived from thecoding sequence of SEQ ID NO:118 shown in FIG. 43.

FIG. 45 shows a nucleotide sequence (SEQ ID NO:126) of a native sequencePRO224 cDNA, wherein SEQ ID NO:126 is a clone designated herein as“DNA33221-1133”.

FIG. 46 shows the amino acid sequence (SEQ ID NO:127) derived from thecoding sequence of SEQ ID NO:126 shown in FIG. 45.

FIG. 47 shows a nucleotide sequence (SEQ ID NO:131) of a native sequencePRO222 cDNA, wherein SEQ ID NO:131 is a clone designated herein as“DNA33107-1135”.

FIG. 48 shows the amino acid sequence (SEQ ID NO:132) derived from thecoding sequence of SEQ ID NO:131 shown in FIG. 47.

FIG. 49 shows a nucleotide sequence (SEQ ID NO:136) of a native sequencePRO234 cDNA, wherein SEQ ID NO:136 is a clone designated herein as“DNA35557-1137”.

FIG. 50 shows the amino acid sequence (SEQ ID NO:137) derived from thecoding sequence of SEQ ID NO:136 shown in FIG. 49.

FIG. 51 shows a nucleotide sequence (SEQ ID NO:141) of a native sequencePRO231 cDNA, wherein SEQ ID NO:141 is a clone designated herein as“DNA34434-1139”.

FIG. 52 shows the amino acid sequence (SEQ ID NO:142) derived from thecoding sequence of SEQ ID NO:141 shown in FIG. 51.

FIG. 53 shows a nucleotide sequence (SEQ ID NO:147) of a native sequencePRO229 cDNA, wherein SEQ ID NO:147 is a clone designated herein as“DNA33100-1159”.

FIG. 54 shows the amino acid sequence (SEQ ID NO:148) derived from thecoding sequence of SEQ ID NO:147 shown in FIG. 53.

FIG. 55 shows a nucleotide sequence (SEQ ID NO:152) of a native sequencePRO238 cDNA, wherein SEQ ID NO:152 is a clone designated herein as“DNA35600-1162”.

FIG. 56 shows the amino acid sequence (SEQ ID NO:153) derived from thecoding sequence of SEQ ID NO:152 shown in FIG. 55.

FIG. 57 shows a nucleotide sequence (SEQ ID NO:158) of a native sequencePRO233 cDNA, wherein SEQ ID NO:158 is a clone designated herein as“DNA34436-1238”.

FIG. 58 shows the amino acid sequence (SEQ ID NO:159) derived from thecoding sequence of SEQ ID NO:158 shown in FIG. 57.

FIG. 59 shows a nucleotide sequence (SEQ ID NO:163) of a native sequencePRO223 cDNA, wherein SEQ ID NO:163 is a clone designated herein as“DNA33206-1165”.

FIG. 60 shows the amino acid sequence (SEQ ID NO:164) derived from thecoding sequence of SEQ ID NO:163 shown in FIG. 59.

FIG. 61 shows a nucleotide sequence (SEQ ID NO:169) of a native sequencePRO235 cDNA, wherein SEQ ID NO:169 is a clone designated herein as“DNA35558-1167”.

FIG. 62 shows the amino acid sequence (SEQ ID NO:170) derived from thecoding sequence of SEQ ID NO:169 shown in FIG. 61.

FIG. 63 shows a nucleotide sequence (SEQ ID NO:174) of a native sequencePRO236 cDNA, wherein SEQ ID NO:174 is a clone designated herein as“DNA35599-1168”.

FIG. 64 shows the amino acid sequence (SEQ ID NO:175) derived from thecoding sequence of SEQ ID NO:174 shown in FIG. 63.

FIG. 65 shows a nucleotide sequence (SEQ ID NO:176) of a native sequencePRO262 cDNA, wherein SEQ ID NO:176 is a clone designated herein as“DNA36992-1168”.

FIG. 66 shows the amino acid sequence (SEQ ID NO:177) derived from thecoding sequence of SEQ ID NO:176 shown in FIG. 65.

FIG. 67 shows a nucleotide sequence (SEQ ID NO:184) of a native sequencePRO239 cDNA, wherein SEQ ID NO:184 is a clone designated herein as“DNA34407-1169”.

FIG. 68 shows the amino acid sequence (SEQ ID NO:185) derived from thecoding sequence of SEQ ID NO:184 shown in FIG. 67.

FIG. 69 shows a nucleotide sequence (SEQ ID NO:189) of a native sequencePRO257 cDNA, wherein SEQ ID NO:189 is a clone designated herein as“DNA35841-1173”.

FIG. 70 shows the amino acid sequence (SEQ ID NO:190) derived from thecoding sequence of SEQ ID NO:189 shown in FIG. 69.

FIG. 71 shows a nucleotide sequence (SEQ ID NO:194) of a native sequencePRO260 cDNA, wherein SEQ ID NO:194 is a clone designated herein as“DNA33470-1175”.

FIG. 72 shows the amino acid sequence (SEQ ID NO:195) derived from thecoding sequence of SEQ ID NO:194 shown in FIG. 71.

FIG. 73 shows a nucleotide sequence (SEQ ID NO:200) of a native sequencePRO263 cDNA, wherein SEQ ID NO:200 is a clone designated herein as“DNA34431-1177”.

FIG. 74 shows the amino acid sequence (SEQ ID NO:201) derived from thecoding sequence of SEQ ID NO:200 shown in FIG. 73.

FIG. 75 shows a nucleotide sequence (SEQ ID NO:206) of a native sequencePRO270 cDNA, wherein SEQ ID NO:206 is a clone designated herein as“DNA39510-1181”.

FIG. 76 shows the amino acid sequence (SEQ ID NO:207) derived from thecoding sequence of SEQ ID NO:206 shown in FIG. 75.

FIG. 77 shows a nucleotide sequence (SEQ ID NO:212) of a native sequencePRO271 cDNA, wherein SEQ ID NO:212 is a clone designated herein as“DNA39423-1182”.

FIG. 78 shows the amino acid sequence (SEQ ID NO:213) derived from thecoding sequence of SEQ ID NO:212 shown in FIG. 77.

FIG. 79 shows a nucleotide sequence (SEQ ID NO:220) of a native sequencePRO272 cDNA, wherein SEQ ID NO:220 is a clone designated herein as“DNA40620-1183”.

FIG. 80 shows the amino acid sequence (SEQ ID NO:221) derived from thecoding sequence of SEQ ID NO:220 shown in FIG. 79.

FIG. 81 shows a nucleotide sequence (SEQ ID NO:226) of a native sequencePRO294 cDNA, wherein SEQ ID NO:226 is a clone designated herein as“DNA40604-1187”.

FIG. 82 shows the amino acid sequence (SEQ ID NO:227) derived from thecoding sequence of SEQ ID NO:226 shown in FIG. 81.

FIG. 83 shows a nucleotide sequence (SEQ ID NO:235) of a native sequencePRO295 cDNA, wherein SEQ ID NO:235 is a clone designated herein as“DNA38268-1188”.

FIG. 84 shows the amino acid sequence (SEQ ID NO:236) derived from thecoding sequence of SEQ ID NO:235 shown in FIG. 83.

FIG. 85 shows a nucleotide sequence (SEQ ID NO:244) of a native sequencePRO293 cDNA, wherein SEQ ID NO:244 is a clone designated herein as“DNA37151-1193”.

FIG. 86 shows the amino acid sequence (SEQ ID NO:245) derived from thecoding sequence of SEQ ID NO:244 shown in FIG. 85.

FIG. 87 shows a nucleotide sequence (SEQ ID NO:249) of a native sequencePRO247 cDNA, wherein SEQ ID NO:249 is a clone designated herein as“DNA35673-1201”.

FIG. 88 shows the amino acid sequence (SEQ ID NO:250) derived from thecoding sequence of SEQ ID NO:249 shown in FIG. 87.

FIG. 89 shows a nucleotide sequence (SEQ ID NO:254) of a native sequencePRO302 cDNA, wherein SEQ ID NO:254 is a clone designated herein as“DNA40370-1217”.

FIG. 90 shows the amino acid sequence (SEQ ID NO:255) derived from thecoding sequence of SEQ ID NO:254 shown in FIG. 89.

FIG. 91 shows a nucleotide sequence (SEQ ID NO:256) of a native sequencePRO303 cDNA, wherein SEQ ID NO:256 is a clone designated herein as“DNA42551-1217”.

FIG. 92 shows the amino acid sequence (SEQ ID NO:257) derived from thecoding sequence of SEQ ID NO:256 shown in FIG. 91.

FIG. 93 shows a nucleotide sequence (SEQ ID NO:258) of a native sequencePRO304 cDNA, wherein SEQ ID NO:258 is a clone designated herein as“DNA39520-1217”.

FIG. 94 shows the amino acid sequence (SEQ ID NO:259) derived from thecoding sequence of SEQ ID NO:258 shown in FIG. 93.

FIG. 95 shows a nucleotide sequence (SEQ ID NO:260) of a native sequencePRO307 cDNA, wherein SEQ ID NO:260 is a clone designated herein as“DNA41225-1217”.

FIG. 96 shows the amino acid sequence (SEQ ID NO:261) derived from thecoding sequence of SEQ ID NO:260 shown in FIG. 95.

FIG. 97 shows a nucleotide sequence (SEQ ID NO:262) of a native sequencePRO343 cDNA, wherein SEQ ID NO:262 is a clone designated herein as“DNA43318-1217”.

FIG. 98 shows the amino acid sequence (SEQ ID NO:263) derived from thecoding sequence of SEQ ID NO:262 shown in FIG. 97.

FIG. 99 shows a nucleotide sequence (SEQ ID NO:284) of a native sequencePRO328 cDNA, wherein SEQ ID NO:284 is a clone designated herein as“DNA40587-1231”.

FIG. 100 shows the amino acid sequence (SEQ ID NO:285) derived from thecoding sequence of SEQ ID NO:284 shown in FIG. 99.

FIG. 101 shows a nucleotide sequence (SEQ ID NO:289) of a nativesequence PRO335 cDNA, wherein SEQ ID NO:289 is a clone designated hereinas “DNA41388-1234”.

FIG. 102 shows the amino acid sequence (SEQ ID NO:290) derived from thecoding sequence of SEQ ID NO:289 shown in FIG. 101.

FIG. 103 shows a nucleotide sequence (SEQ ID NO:291) of a nativesequence PRO331 cDNA, wherein SEQ ID NO:291 is a clone designated hereinas “DNA40981-1234”.

FIG. 104 shows the amino acid sequence (SEQ ID NO:292) derived from thecoding sequence of SEQ ID NO:291 shown in FIG. 103.

FIG. 105 shows a nucleotide sequence (SEQ ID NO:293) of a nativesequence PRO326 cDNA, wherein SEQ ID NO:293 is a clone designated hereinas “DNA37140-1234”.

FIG. 106 shows the amino acid sequence (SEQ ID NO:294) derived from thecoding sequence of SEQ ID NO:293 shown in FIG. 105.

FIG. 107 shows a nucleotide sequence (SEQ ID NO:309) of a nativesequence PRO332 cDNA, wherein SEQ ID NO:309 is a clone designated hereinas “DNA40982-1235”.

FIG. 108 shows the amino acid sequence (SEQ ID NO:310) derived from thecoding sequence of SEQ ID NO:309 shown in FIG. 107.

FIG. 109 shows a nucleotide sequence (SEQ ID NO:314) of a nativesequence PRO334 cDNA, wherein SEQ ID NO:314 is a clone designated hereinas “DNA41379-1236”.

FIG. 110 shows the amino acid sequence (SEQ ID NO:315) derived from thecoding sequence of SEQ ID NO:314 shown in FIG. 109.

FIG. 111 shows a nucleotide sequence (SEQ ID NO:319) of a nativesequence PRO346 cDNA, wherein SEQ ID NO:319 is a clone designated hereinas “DNA44167-1243”.

FIG. 112 shows the amino acid sequence (SEQ ID NO:320) derived from thecoding sequence of SEQ ID NO:319 shown in FIG. 111.

FIG. 113 shows a nucleotide sequence (SEQ ID NO:324) of a nativesequence PRO268 cDNA, wherein SEQ ID NO:324 is a clone designated hereinas “DNA39427-1179”.

FIG. 114 shows the amino acid sequence (SEQ ID NO:325) derived from thecoding sequence of SEQ ID NO:324 shown in FIG. 113.

FIG. 115 shows a nucleotide sequence (SEQ ID NO:331) of a nativesequence PRO330 cDNA, wherein SEQ ID NO:331 is a clone designated hereinas “DNA40603-1232”.

FIG. 116 shows the amino acid sequence (SEQ ID NO:332) derived from thecoding sequence of SEQ ID NO:331 shown in FIG. 115.

FIG. 117 shows a nucleotide sequence (SEQ ID NO:338) of a nativesequence PRO339 cDNA, wherein SEQ ID NO:338 is a clone designated hereinas “DNA43466-1225”.

FIG. 118 shows the amino acid sequence (SEQ ID NO:339) derived from thecoding sequence of SEQ ID NO:338 shown in FIG. 117.

FIG. 119 shows a nucleotide sequence (SEQ ID NO:340) of a nativesequence PRO310 cDNA, wherein SEQ ID NO:340 is a clone designated hereinas “DNA43046-1225”.

FIG. 120 shows the amino acid sequence (SEQ ID NO:341) derived from thecoding sequence of SEQ ID NO:340 shown in FIG. 119.

FIG. 121 shows a nucleotide sequence (SEQ ID NO:376) of a nativesequence PRO244 cDNA, wherein SEQ ID NO:376 is a clone designated hereinas “DNA35668-1171”.

FIG. 122 shows the amino acid sequence (SEQ ID NO:377) derived from thecoding sequence of SEQ ID NO:376 shown in FIG. 121.

FIG. 123 shows a nucleotide sequence (SEQ ID NO:422) of a nativesequence PRO1868 cDNA, wherein SEQ ID NO:422 is a clone designatedherein as “DNA77624-2515”.

FIG. 124 shows the amino acid sequence (SEQ ID NO:423) derived from thecoding sequence of SEQ ID NO:422 shown in FIG. 123.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Definitions

The terms “PRO polypeptide” and “PRO” as used herein and whenimmediately followed by a numerical designation refer to variouspolypeptides, wherein the complete designation (i.e., PRO/number) refersto specific polypeptide sequences as described herein. The terms“PRO/number polypeptide” and “PRO/number” wherein the term “number” isprovided as an actual numerical designation as used herein encompassnative sequence polypeptides and polypeptide variants (which are furtherdefined herein). The PRO polypeptides described herein may be isolatedfrom a variety of sources, such as from human tissue types or fromanother source, or prepared by recombinant or synthetic methods.

A “native sequence PRO polypeptide” comprises a polypeptide having thesame amino acid sequence as the corresponding PRO polypeptide derivedfrom nature. Such native sequence PRO polypeptides can be isolated fromnature or can be produced by recombinant or synthetic means. The term“native sequence PRO polypeptide” specifically encompassesnaturally-occurring truncated or secreted forms of the specific PROpolypeptide (e.g., an extracellular domain sequence),naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of the polypeptide. In variousembodiments of the invention, the native sequence PRO polypeptidesdisclosed herein are mature or full-length native sequence polypeptidescomprising the full-length amino acids sequences shown in theaccompanying figures. Start and stop codons are shown in bold font andunderlined in the figures. However, while the PRO polypeptide disclosedin the accompanying figures are shown to begin with methionine residuesdesignated herein as amino acid position 1 in the figures, it isconceivable and possible that other methionine residues located eitherupstream or downstream from the amino acid position 1 in the figures maybe employed as the starting amino acid residue for the PRO polypeptides.

The PRO polypeptide “extracellular domain” or “ECD” refers to a form ofthe PRO polypeptide which is essentially free of the transmembrane andcytoplasmic domains. Ordinarily, a PRO polypeptide ECD will have lessthan 1% of such transmembrane and/or cytoplasmic domains and preferably,will have less than 0.5% of such domains. It will be understood that anytransmembrane domains identified for the PRO polypeptides of the presentinvention are identified pursuant to criteria routinely employed in theart for identifying that type of hydrophobic domain. The exactboundaries of a transmembrane domain may vary but most likely by no morethan about 5 amino acids at either end of the domain as initiallyidentified herein. Optionally, therefore, an extracellular domain of aPRO polypeptide may contain from about 5 or fewer amino acids on eitherside of the transmembrane domain/extracellular domain boundary asidentified in the Examples or specification and such polypeptides, withor without the associated signal peptide, and nucleic acid encodingthem, are comtemplated by the present invention.

The approximate location of the “signal peptides” of the various PROpolypeptides disclosed herein are shown in the present specificationand/or the accompanying figures. It is noted, however, that theC-terminal boundary of a signal peptide may vary, but most likely by nomore than about 5 amino acids on either side of the signal peptideC-terminal boundary as initially identified herein, wherein theC-terminal boundary of the signal peptide may be identified pursuant tocriteria routinely employed in the art for identifying that type ofamino acid sequence element (e.g., Nielsen et al., Prot. Eng. 10: 1-6(1997) and von Heinje et al., Nucl. Acids. Res. 14:4683-4690 (1986)).Moreover, it is also recognized that, in some cases, cleavage of asignal sequence from a secreted polypeptide is not entirely uniform,resulting in more than one secreted species. These mature polypeptides,where the signal peptide is cleaved within no more than about 5 aminoacids on either side of the C-terminal boundary of the signal peptide asidentified herein, and the polynucleotides encoding them, arecontemplated by the present invention.

“PRO polypeptide variant” means an active PRO polypeptide as definedabove or below having at least about 80% amino acid sequence identitywith a full-length native sequence PRO polypeptide sequence as disclosedherein, a PRO polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a PRO polypeptide, with orwithout the signal peptide, as disclosed herein or any other fragment ofa full-length PRO polypeptide sequence as disclosed herein. Such PROpolypeptide variants include, for instance, PRO polypeptides wherein oneor more amino acid residues are added, or deleted, at the N- orC-terminus of the full-length native amino acid sequence. Ordinarily, aPRO polypeptide variant will have at least about 80% amino acid sequenceidentity, preferably at least about 81% amino acid sequence identity,more preferably at least about 82% amino acid sequence identity, morepreferably at least about 83% amino acid sequence identity, morepreferably at least about 84% amino acid sequence identity, morepreferably at least about 85% amino acid sequence identity, morepreferably at least about 86% amino acid sequence identity, morepreferably at least about 87% amino acid sequence identity, morepreferably at least about 88% amino acid sequence identity, morepreferably at least about 89% amino acid sequence identity, morepreferably at least about 90% amino acid sequence identity, morepreferably at least about 91% amino acid sequence identity, morepreferably at least about 92% amino acid sequence identity, morepreferably at least about 93% amino acid sequence identity, morepreferably at least about 94% amino acid sequence identity, morepreferably at least about 95% amino acid sequence identity, morepreferably at least about 96% amino acid sequence identity, morepreferably at least about 97% amino acid sequence identity, morepreferably at least about 98% amino acid sequence identity and mostpreferably at least about 99% amino acid sequence identity with afull-length native sequence PRO polypeptide sequence as disclosedherein, a PRO polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a PRO polypeptide, with orwithout the signal peptide, as disclosed herein or any otherspecifically defined fragment of a full-length PRO polypeptide sequenceas disclosed herein. Ordinarily, PRO variant polypeptides are at leastabout 10 amino acids in length, often at least about 20 amino acids inlength, more often at least about 30 amino acids in length, more oftenat least about 40 amino acids in length, more often at least about 50amino acids in length, more often at least about 60 amino acids inlength, more often at least about 70 amino acids in length, more oftenat least about 80 amino acids in length, more often at least about 90amino acids in length, more often at least about 100 amino acids inlength, more often at least about 150 amino acids in length, more oftenat least about 200 amino acids in length, more often at least about 300amino acids in length, or more.

“Percent (%) amino acid sequence identity” with respect to the PROpolypeptide sequence s identified herein is defined as the percentage ofamino acid residues in a candidate sequence that are identical with theamino acid residues in the specific PRO polypeptide sequence, afteraligning the sequences and introducing gaps, if necessary, t o achievethe maximum percent sequence id entity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the artcan determine appropriate parameters for measuring alignment, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared. For purposes herein, however, % aminoacid sequence identity values are generated using the sequencecomparison computer program ALIGN-2, wherein the complete source codefor the ALIGN-2 program is provided in Table 1 below. The ALIGN-2sequence comparison computer program was authored by Genentech, Inc. andthe source code shown in Table 1 below has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin Table 1 below. The ALIGN-2 program should be compiled for use on aUNIX operating system, preferably digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction {fraction (X/Y)}where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. As examples of % amino acid sequence identitycalculations using this method, Tables 2 and 3 demonstrate how tocalculate the % amino acid sequence identity of the amino acid sequencedesignated “Comparison Protein” to the amino acid sequence designated“PRO”, wherein “PRO” represents the amino acid sequence of ahypothetical PRO polypeptide of interest, “Comparison Protein”represents the amino acid sequence of a polypeptide against which the“PRO” polypeptide of interest is being compared, and “X, “Y” and “Z”each represent different hypothetical amino acid residues.

Unless specifically stated otherwise, all % amino acid sequence identityvalues used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program. However, % aminoacid sequence identity values may also be obtained as described below byusing the WU-BLAST-2 computer program (Altschul et al., Methods inEnzymology 266:460-480 (1996)). Most of the WU-BLAST-2 search parametersare set to the default values. Those not set to default values, i.e.,the adjustable parameters, are set with the following values: overlapspan=1, overlap fraction=0.125, word threshold (T)=11, and scoringmatrix=BLOSUM62. When WU-BLAST-2 is employed, a % amino acid sequenceidentity value is determined by dividing (a) the number of matchingidentical amino acid residues between the amino acid sequence of the PROpolypeptide of interest having a sequence derived from the native PROpolypeptide and the comparison amino acid sequence of interest (i.e.,the sequence against which the PRO polypeptide of interest is beingcompared which may be a PRO variant polypeptide) as determined byWU-BLAST-2 by (b) the total number of amino acid residues of the PROpolypeptide of interest. For example, in the statement “a polypeptidecomprising an the amino acid sequence A which has or having at least 80%amino acid sequence identity to the amino acid sequence B”, the aminoacid sequence A is the comparison amino acid sequence of interest andthe amino acid sequence B is the amino acid sequence of the PROpolypeptide of interest.

Percent amino acid sequence identity may also be determined using thesequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic AcidsRes. 25:3389-3402 (1997)). NCBI-BLAST2 uses several search parameters,wherein all of those search parameters are set to default valuesincluding, for example, unmask=yes, strand=all, expected occurrences=10,minimum low complexity length=15/5, multi-pass e-value=0.01, constantfor multi-pass=25, dropoff for final gapped alignment=25 and scoringmatrix=BLOSUM62.

In situations where NCBI-BLAST2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction {fraction (X/Y)}where X is the number of amino acid residues scored as identical matchesby the sequence alignment program NCBI-BLAST2 in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A.

“PRO variant polynucleotide” or “PRO variant nucleic acid sequence”means a nucleic acid molecule which encodes an active PRO polypeptide asdefined below and which has at least about 80% nucleic acid sequenceidentity with a nucleotide acid sequence encoding a full-length nativesequence PRO polypeptide sequence as disclosed herein, a full-lengthnative sequence PRO polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a PRO polypeptide, with orwithout the signal peptide, as disclosed herein or any other fragment ofa full-length PRO polypeptide sequence as disclosed herein. Ordinarily,a PRO variant polynucleotide will have at least about 80% nucleic acidsequence identity, more preferably at least about 81% nucleic acidsequence identity, more preferably at least about 82% nucleic acidsequence identity, more preferably at least about 83% nucleic acidsequence identity, more preferably at least about 84% nucleic acidsequence identity, more preferably at least about 85% nucleic acidsequence identity, more preferably at least about 86% nucleic acidsequence identity, more preferably at least about 87% nucleic acidsequence identity, more preferably at least about 88% nucleic acidsequence identity, more preferably at least about 89% nucleic acidsequence identity, more preferably at least about 90% nucleic acidsequence identity, more preferably at least about 91% nucleic acidsequence identity, more preferably at least about 92% nucleic acidsequence identity, more preferably at least about 93% nucleic acidsequence identity, more preferably at least about 94% nucleic acidsequence identity, more preferably at least about 95% nucleic acidsequence identity, more preferably at least about 96% nucleic acidsequence identity, more preferably at least about 97% nucleic acidsequence identity, more preferably at least about 98% nucleic acidsequence identity and yet more preferably at least about 99% nucleicacid sequence identity with a nucleic acid sequence encoding afull-length native sequence PRO polypeptide sequence as disclosedherein, a full-length native sequence PRO polypeptide sequence lackingthe signal peptide as disclosed herein, an extracellular domain of a PROpolypeptide, with or without the signal sequence, as disclosed herein orany other fragment of a full-length PRO polypeptide sequence asdisclosed herein. Variants do not encompass the native nucleotidesequence.

Ordinarily, PRO variant polynucleotides are at least about 30nucleotides in length, often at least about 60 nucleotides in length,more often at least about 90 nucleotides in length, more often at leastabout 120 nucleotides in length, more often at least about 150nucleotides in length, more often at least about 180 nucleotides inlength, more often at least about 210 nucleotides in length, more oftenat least about 240 nucleotides in length, more often at least about 270nucleotides in length, more often at least about 300 nucleotides inlength, more often at least about 450 nucleotides in length, more oftenat least about 600 nucleotides in length, more often at least about 900nucleotides in length, or more.

“Percent (%) nucleic acid sequence identity” with respect toPRO-encoding nucleic acid sequences identified herein is defined as thepercentage of nucleotides in a candidate sequence that are identicalwith the nucleotides in the PRO nucleic acid sequence of interest, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity. Alignment for purposes ofdetermining percent nucleic acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. For purposes herein, however, % nucleicacid sequence identity values are generated using the sequencecomparison computer program ALIGN-2, wherein the complete source codefor the ALIGN-2 program is provided in Table 1 below. The ALIGN-2sequence comparison computer program was authored by Genentech, Inc. andthe source code shown in Table 1 below has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin Table 1 below. The ALIGN-2 program should be compiled for use on aUNIX operating system, preferably digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for nucleic acid sequencecomparisons, the % nucleic acid sequence identity of a given nucleicacid sequence C to, with, or against a given nucleic acid sequence D(which can alternatively be phrased as a given nucleic acid sequence Cthat has or comprises a certain % nucleic acid sequence identity to,with, or against a given nucleic acid sequence D) is calculated asfollows:100 times the fraction {fraction (W/Z)}where W is the number of nucleotides scored as identical matches by thesequence alignment program ALIGN-2 in that program's alignment of C andD, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C. As examples of % nucleic acid sequence identitycalculations, Tables 4 and 5, demonstrate how to calculate the % nucleicacid sequence identity of the nucleic acid sequence designated“Comparison DNA” to the nucleic acid sequence designated “PRO-DNA”,wherein “PRO-DNA” represents a hypothetical PRO-encoding nucleic acidsequence of interest, “Comparison DNA” represents the nucleotidesequence of a nucleic acid molecule against which the “PRO-DNA” nucleicacid molecule of interest is being compared, and “N”, “L” and “V” eachrepresent different hypothetical nucleotides.

Unless specifically stated otherwise, all % nucleic acid sequenceidentity values used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program. However, %nucleic acid sequence identity values may also be obtained as describedbelow by using the WU-BLAST-2 computer program (Altschul et al., Methodsin Enzymology 266:460480 (1996)). Most of the WU-BLAST-2 searchparameters are set to the default values. Those not set to defaultvalues, i.e., the adjustable parameters, are set with the followingvalues: overlap span=1, overlap fraction=0.125, word threshold (T)=11,and scoring matrix=BLOSUM62. When WU-BLAST-2 is employed, a % nucleicacid sequence identity value is determined by dividing (a) the number ofmatching identical nucleotides between the nucleic acid sequence of thePRO polypeptide-encoding nucleic acid molecule of interest having asequence derived from the native sequence PRO polypeptide-encodingnucleic acid and the comparison nucleic acid molecule of interest (i.e.,the sequence against which the PRO polypeptide-encoding nucleic acidmolecule of interest is being compared which may be a variant PROpolynucleotide) as determined by WU-BLAST-2 by (b) the total number ofnucleotides of the PRO polypeptide-encoding nucleic acid molecule ofinterest. For example, in the statement “an isolated nucleic acidmolecule comprising a nucleic acid sequence A which has or having atleast 80% nucleic acid sequence identity to the nucleic acid sequenceB”, the nucleic acid sequence A is the comparison nucleic acid moleculeof interest and the nucleic acid sequence B is the nucleic acid sequenceof the PRO polypeptide-encoding nucleic acid molecule of interest.

Percent nucleic acid sequence identity may also be determined using thesequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic AcidsRes. 25:3389-3402 (1997)). NCBI-BLAST2 uses several search parameters,wherein all of those search parameters are set to default valuesincluding, for example, unmask=yes, strand=all, expected occurrences=10,minimum low complexity length=15/5, multi-pass e-value=0.01, constantfor multi-pass=25, dropoff for final gapped alignment=25 and scoringmatrix=BLOSUM62.

In situations where NCBI-BLAST2 is employed for sequence comparisons,the % nucleic acid sequence identity of a given nucleic acid sequence Cto, with, or against a given nucleic acid sequence D (which canalternatively be phrased as a given nucleic acid sequence C that has orcomprises a certain % nucleic acid sequence identity to, with, oragainst a given nucleic acid sequence D) is calculated as follows:100 times the fraction {fraction (W/Z)}where W is the number of nucleotides scored as identical matches by thesequence alignment program NCBI-BLAST2 in that program's alignment of Cand D, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C.

In other embodiments, PRO variant polynucleotides are nucleic acidmolecules that encode an active PRO polypeptide and which are capable ofhybridizing, preferably under stringent hybridization and washconditions, to nucleotide sequences encoding a full-length PROpolypeptide as disclosed herein. PRO variant polypeptides may be thosethat are encoded by a PRO variant polynucleotide.

The term “positives”, in the context of sequence comparison performed asdescribed above, includes residues in the sequences compared that arenot identical but have similar properties (e.g. as a result ofconservative substitutions, see Table 6 below). For purposes herein, the% value of positives is determined by dividing (a) the number of aminoacid residues scoring a positive value between the PRO polypeptide aminoacid sequence of interest having a sequence derived from the native PROpolypeptide sequence and the comparison amino acid sequence of interest(i.e., the amino acid sequence against which the PRO polypeptidesequence is being compared) as determined in the BLOSUM62 matrix ofWU-BLAST-2 by (b) the total number of amino acid residues of the PROpolypeptide of interest.

Unless specifically stated otherwise, the % value of positives iscalculated as described in the immediately preceding paragraph. However,in the context of the amino acid sequence identity comparisons performedas described for ALIGN-2 and NCBI-BLAST-2 above, includes amino acidresidues in the sequences compared that are not only identical, but alsothose that have similar properties. Amino acid residues that score apositive value to an amino acid residue of interest are those that areeither identical to the amino acid residue of interest or are apreferred substitution (as defined in Table 6 below) of the amino acidresidue of interest.

For amino acid sequence comparisons using ALIGN-2 or NCBI-BLAST2, the %value of positives of a given amino acid sequence A to, with, or againsta given amino acid sequence B (which can alternatively be phrased as agiven amino acid sequence A that has or comprises a certain % positivesto, with, or against a given amino acid sequence B) is calculated asfollows:100 times the fraction {fraction (X/Y)}where X is the number of amino acid residues scoring a positive value asdefined above by the sequence alignment program ALIGN-2 or NCBI-BLAST2in that program's alignment of A and B, and where Y is the total numberof amino acid residues in B. It will be appreciated that where thelength of amino acid sequence A is not equal to the length of amino acidsequence B, the % positives of A to B will not equal the % positives ofB to A.

“Isolated,” when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the PRO polypeptidenatural environment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

An “isolated” PRO polypeptide-encoding nucleic acid or otherpolypeptide-encoding nucleic acid is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe polypeptide-encoding nucleic acid. An isolated polypeptide-encodingnucleic acid molecule is other than in the form or setting in which itis found in nature. Isolated polypeptide-encoding nucleic acid moleculestherefore are distinguished from the specific polypeptide-encodingnucleic acid molecule as it exists in natural cells. However, anisolated polypeptide-encoding nucleic acid molecule includespolypeptide-encoding nucleic acid molecules contained in cells thatordinarily express the polypeptide where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The term “antibody” is used in the broadest sense and specificallycovers, for example, single anti-PRO monoclonal antibodies (includingagonist, antagonist, and neutralizing antibodies), anti-PRO antibodycompositions with polyepitopic specificity, single chain anti-PROantibodies, and fragments of anti-PRO antibodies (see below). The term“monoclonal antibody” as used herein refers to an antibody obtained froma population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally-occurring mutations that may be present in minoramounts.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodiumphosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5× SSC (0.75 M NaCl, 0.075 M sodium citrate), 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2× SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1× SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and %SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5× SSC (150 mM NaCl, 15 mM trisodiumcitrate),50 mM sodiumphosphate (pH 7.6), 5× Denhardt's solution, 10% dextransulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1× SSC at about 37-50° C. The skilled artisanwill recognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising a PRO polypeptide fused to a “tag polypeptide”.The tag polypeptide has enough residues to provide an epitope againstwhich an antibody can be made, yet is short enough such that it does notinterfere with activity of the polypeptide to which it is fused. The tagpolypeptide preferably also is fairly unique so that the antibody doesnot substantially cross-react with other epitopes. Suitable tagpolypeptides generally have at least six amino acid residues and usuallybetween about 8 and 50 amino acid residues (preferably, between about 10and 20 amino acid residues).

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

“Active” or “activity” for the purposes herein refers to form(s) of aPRO polypeptide which retain a biological and/or an immunologicalactivity of native or naturally-occurring PRO, wherein “biological”activity refers to a biological function (either inhibitory orstimulatory) caused by a native or naturally-occurring PRO other thanthe ability to induce the production of an antibody against an antigenicepitope possessed by a native or naturally-occurring PRO and an“immunological” activity refers to the ability to induce the productionof an antibody against an antigenic epitope possessed by a native ornaturally-occurring PRO.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a native PRO polypeptide disclosed herein. In asimilar manner, the term “agonist” is used in the broadest sense andincludes any molecule that mimics a biological activity of a native PROpolypeptide disclosed herein. Suitable agonist or antagonist moleculesspecifically include agonist or antagonist antibodies or antibodyfragments, fragments or amino acid sequence variants of native PROpolypeptides, peptides, antisense oligonucleotides, small organicmolecules, etc. Methods for identifying agonists or antagonists of a PROpolypeptide may comprise contacting a PRO polypeptide with a candidateagonist or antagonist molecule and measuring a detectable change in oneor more biological activities normally associated with the PROpolypeptide.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder. Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats,rabbits, etc. Preferably, the mammal is human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.8(10): 1057-1062 [1995]); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, a designation reflecting the abilityto crystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site. The Fab fragment alsocontains the constant domain of the light chain and the first constantdomain (CH1) of the heavy chain. Fab fragments differ from Fab′fragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CH1 domain including one or more cysteines from theantibody hinge region. Fab′-SH is the designation herein for Fab′ inwhich the cysteine residue(s) of the constant domains bear a free thiolgroup. F(ab′)₂ antibody fragments originally were produced as pairs ofFab′ fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa and lambda, based on the amino acid sequences of their constantdomains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding. For a review ofsFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93111161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibodyso as to generate a “labeled” antibody. The label may be detectable byitself (e.g. radioisotope labels or fluorescent labels) or, in the caseof an enzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable.

By “solid phase” is meant a non-aqueous matrix to which the antibody ofthe present invention can adhere. Examples of solid phases encompassedherein include those formed partially or entirely of glass (e.g.,controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g., an affinity chromatography column). This term alsoincludes a discontinuous solid phase of discrete particles, such asthose described in U.S. Pat. No. 4,275,149.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as a PRO polypeptide or antibody thereto) to a mammal. Thecomponents of the liposome are commonly arranged in a bilayer formation,similar to the lipid arrangement of biological membranes.

A “small molecule” is defined herein to have a molecular weight belowabout 500 Daltons.

“PRO317-associated disorder” refers to a pathological condition ordisease wherein PRO317 is over- or underexpressed. Such disordersinclude diseases of the female genital tract or of the endometrium of amammal, including hyperplasia, endometritis, endometriosis, wherein thepatient is at risk for infertility due to endometrial factor,endometrioma, and endometrial cancer, especially those diseasesinvolving abnormal bleeding such as a gynecological disease. They alsoinclude diseases involving angiogenesis, wherein the angiogenesisresults in a pathological condition, such as cancer involving solidtumors (the therapy for the disorder would result in decreasedvascularization and a decline in growth and metastasis of a variety oftumors). Alternatively, the angiogenesis may be beneficial, such as forischemia, especially coronary ischemia. Hence, these disorders includethose found in patients whose hearts are functioning but who have ablocked blood supply due to atherosclerotic coronary artery disease, andthose with a functioning but underperfused heart, including patientswith coronary arterial disease who are not optimal candidates forangioplasty and coronary artery by-pass surgery. The disorders alsoinclude diseases involving the kidney or originating from the kidneytissue, such as polycystic kidney disease and chronic and acute renalfailure.

TABLE 1 /*  *  * C-C increased from 12 to 15  * Z is average of EQ  * Bis average of ND  * match with stop is _M; stop-stop = 0; J (joker)match = 0  */ #define _M −8 /* value of a match with a stop */ int_day[26][26] = { /*  A B C D E F G H I J K L M N O P Q R S T U V W X Y Z*/ /* A */ { 2, 0, −2, 0, 0, −4, 1, −1, −1, 0, −1, −2, −1, 0, _M, 1, 0,−2, 1, 1, 0, 0, −6, 0, −3, 0}, /* B */ { 0, 3, −4, 3, 2, −5, 0, 1, −2,0, 0, −3, −2, 2, _M, −1, 1, 0, 0, 0, 0, −2, −5, 0, −3, 1}, /* C */ {−2,−4, 15, −5, −5, −4, −3, −3, −2, 0, −5, −6, −5, −4, _M, −3, −5, −4, 0,−2, 0, −2, −8, 0, 0, −5}, /* D */ {0, 3, −5, 4, 3, −6, 1, 1, −2, 0, 0,−4, −3, 2, _M, −1, 2, −1, 0, 0, 0, −2, −7, 0, −4, 2}, /* E */ {0, 2, −5,3, 4, −5, 0, 1, −2, 0, 0, −3, −2, 1, _M, −1, 2, −1, 0, 0, 0, −2, −7, 0,−4, 3}, /* F */ {−4, −5, −4, −6, −5, 9, −5, −2, 1, 0, −5, 2, 0, −4, _M,−5, −5, −4, −3, −3, 0, −1, 0, 0, 7, −5}, /* G */ { 1, 0, −3, 1, 0, −5,5, −2, −3, 0, −2, −4, −3, 0, _M, −1, −1, −3, 1, 0, 0, −1, −7, 0, −5, 0},/* H */ {−1, 1, −3, 1, 1, −2, −2, 6, −2, 0, 0, −2, −2, 2, _M, 0, 3, 2,−1, −1, 0, −2, −3, 0, 0, 2}, /* I */ {−1, −2, −2, −2, −2, 1, −3, −2, 5,0, −2, 2, 2, −2, _M, −2, −2, −2, −1, 0, 0, 4, −5, 0, −1, −2}, /* J */{0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, _M, 0, 0, 0, 0, 0, 0, 0, 0,0, 0, 0}, /* K */ {−1, 0, −5, 0, 0, −5, −2, 0, −2, 0, 5, −3, 0, 1, _M,−1, 1, 3, 0, 0, 0, −2, −3, 0, −4, 0}, /* L */ {−2, −3, −6, −4, −3, 2,−4, −2, 2, 0, −3, 6, 4, −3, _M, −3, −2, −3, −3 , −1, 0, 2, −2, 0, −1,−2} /* M */ {−1, −2, −5, −3, −2, 0, −3, −2, 2, 0, 0, 4, 6, −2, _M, −2,−1, 0, −2, −1, 0, 2, −4, 0, −2, −1}, /* N */ {0, 2, −4, 2, 1, −4, 0, 2,−2, 0, 1, −3, −2, 2, _M, −1, 1, 0, 1, 0, 0, −2, −4, 0, −2, 1}, /* O */{_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,0,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,}, /* P */ {1, −1, −3, −1, −1, −5,−1, 0, −2, 0, −1, −3, −2, −1,_M, 6, 0, 0, 1, 0, 0, −1, −6, 0, −5, 0}, /*Q */ {0, 1, −5, 2, 2, −5, −1, 3, −2, 0, 1, −2, −1, 1, _M, 0, 4, 1, −1,−1, 0, −2, −5, 0, −4, 3}, /* R */ {−2, 0, −4, −1, −1, −4, −3, 2, −2, 0,3, −3, 0, 0, _M, 0, 1, 6, 0, −1, 0, −2, 2, 0, −4, 0}, /* S */ { 1, 0, 0,0, 0, −3, 1, −1, −1, 0, 0, −3, −2, 1, _M, 1, −1, 0, 2, 1, 0, −1, −2, 0,−3, 0}, /* T */ { 1, 0, −2, 0, 0, −3, 0, −1, 0, 0, 0, −1, −1, 0, _M, 0,−1, −1, 1, 3, 0, 0, −5, 0, −3, 0}, /* U */ {0, 0, 0, 0, 0, 0, 0, 0, 0,0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, /* V */ {0, −2, −2,−2, −2, −1, −1, −2, 4, 0, −2, 2, 2, −2,_M, −1, −2, −2, −1, 0, 0, 4, −6,0, −2, −2}, /* W */ {−6, −5, −8, −7, −7, 0, −7, −3, −5, 0, −3, −2, −4,−4,_M, −6, −5, 2, −2, −5, 0, −6, 17, 0, 0, −6}, /* X */ {0, 0, 0, 0, 0,0, 0, 0, 0, 0, 0, 0, 0, 0, _M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, /* Y */{−3, −3, 0, −4, −4, 7, −5, 0, −1, 0, −4, −1, −2, −2, _M, −5, −4, −4, −3,−3, 0, −2, 0, 0, 10, −4}, /* Z */ { 0, 1, −5, 2, 3, −5, 0, 2, −2, 0, 0,−2, −1, 1,_M, 0, 3, 0, 0, 0, 0, −2, −6, 0, −4, 4}, }; /*  */ #include<stdio.h> #include <ctype.h> #define MAXJMP  16 /* max jumps in a diag*/ #define MAXGAP  24 /* don't continue to penalize gaps larger thanthis */ #define JMPS 1024 /* max jmps in an path */ #define MX   4 /*save if there's at least MX-1 bases since last jmp */ #define DMAT   3/* value of matching bases */ #define DMIS   0 /* penalty for mismatchedbases */ #define DINS0   8 /* penalty for a gap */ #define DINS1   1 /*penalty per base */ #define PINS0   8 /* penalty for a gap */ #definePINS1   4 /* penalty per residue */ struct jmp { short n[MAXJMP]; /*size of jmp (neg for dely) */ unsigned short x[MAXJMP]; /* base no. ofjmp in seq x */ /* limits seq to 2{circumflex over ( )}16 −1 */ };struct diag { int score; /* score at last jmp */ long offset; /* offsetof prev block */ short ijmp; /* current jmp index */ struct jmp jp; /*list of jmps */ }; struct path { int spc; /* number of leading spaces */short n[JMPS]; /* size of jmp (gap) */ int x[JMPS]; /* loc of jmp (lastelem before gap) */ }; char *ofile; /* output file name */ char*namex[2]; /* seq names: getseqs() */ char *prog; /* prog name for errmsgs */ char *seqx[2];   /* seqs: getseqs() */ int dmax; /* best diag:nw() */ int dmax0; /* final diag */ int dna; /* set if dna: main() */int endgaps; /* set if penalizing end gaps */ int gapx, gapy; /* totalgaps in seqs */ int len0, len1; /* seq lens */ int ngapx, ngapy; /*total size of gaps */ int smax; /* max score: nw() */ int *xbm; /*bitmap for matching */ long offset; /* current offset in jmp file */struct diag *dx; /* holds diagonals */ struct path pp[2]; /* holds pathfor seqs */ char *calloc(), *malloc(), *index(), *strcpy(); char*getseq(), *g_calloc(); /* Needleman-Wunsch alignment program  *  *usage: progs file1 file2  * where file1 and file2 are two dna or twoprotein sequences.  * The sequences can be in upper- or lower-case anmay contain ambiguity  * Any lines beginning with ‘;’, ‘>’ or ‘<’ areignored  * Max file length is 65535 (limited by unsigned short x in thejmp struct)  * A sequence with ⅓ or more of its elements ACGTU isassumed to be DNA  * Output is in the file “align.out”  *  * The programmay create a tmp file in /tmp to hold info about traceback.  * Originalversion developed under BSD 4.3 on a vax 8650  */ #include “nw.h”#include “day.h” static _dbval[26] = {1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0 }; static_pbval[26] = { 1, 2|(1< <(‘D’-‘A’))|(1< <(‘N’-‘A’)), 4, 8, 16, 32, 64,128, 256, 0×FFFFFFF, 1< <10, 1< <11, 1< <12, 1< <13, 1< <14, 1< <15, 1<<16, 1< <17, 1< <18, 1< <19, 1< <20, 1< <21, 1< <22, 1< <23, 1< <24, 1<<25|(1< <(‘E’-‘A’))|(1< <(‘Q’-‘A’)) }; main(ac, av) main int ac; char*av[]; { prog = av[0]; if(ac != 3) { fprintf(stderr, “usage: %s file1file2\n”, prog); fprintf(stderr, “where file1 and file2 are two dna ortwo protein sequences.\n”); fprintf(stderr, “The sequences can be inupper- or lower-case\n”); fprintf(stderr, “Any lines beginning with ‘;’or ‘<’ are ignored\n”); fprintf(stderr, “Output is in the file\“align.out\”\n”); exit(1); } namex[0] = av[1]; namex[1] = av[2];seqx[0] = getseq(namex[0], &len0); seqx[1] = getseq(namex[1], &len1);xbm = (dna)? _dbval : _pbval; endgaps = 0; /* 1 to penalize endgaps */ofile = “align.out”; /* output file */ nw(); /* fill in the matrix, getthe possible jmps */ readjmps(); /* get the actual jmps */ print(); /*print stats, alignment */ cleanup(0); /* unlink any tmp files */ } /* dothe alignment, return best score: main()  * dna: values in Fitch andSmith, PNAS, 80, 1382-1386, 1983  * pro: PAM 250 values  * When scoresare equal, we prefer mismatches to any gap, prefer  * a new gap toextending an ongoing gap, and prefer a gap in seqx  * to a gap in seq y. */ nw() nw { char *px, *py;   /* seqs and ptrs */ int *ndely, *dely; /*keep track of dely */ int ndelx, delx; /* keep track of delx */ int*tmp; /* for swapping row0, row1 */ int mis; /* score for each type */int ins0, ins1; /* insertion penalties */ register id; /* diagonal index*/ register ij; /* jmp index */ register *col0, *col1; /* score forcurr, last row */ register xx, yy; /* index into seqs */ dx = (structdiag *)g_calloc(“to get diags”, len0+len1+1, sizeof(struct diag)); ndely= (int *)g_calloc(“to get ndely”, len1+1, sizeof(int)); dely = (int*)g_calloc(“to get dely”, len1+1, sizeof(int)); col0 = (int*)g_calloc(“to get col0”, len1+1, sizeof(int)); col1 = (int*)g_calloc(“to get col1”, len1+1, sizeof(int)); ins0 = (dna)? DINS0 :PINS0; ins1 = (dna)? DINS1 : PlNS1; smax = −10000; if (endgaps) { for(col0[0] = dely[0] = −ins0, yy = 1; yy <= len1; yy++) { col0[yy] =dely[yy] = col0[yy−1] − ins1; ndely[yy] = yy; } col0[0] = 0; /* WatermanBull Math Biol 84 */ } else for (yy= 1; yy <= len1; yy++) dely[yy] =−ins0; /* fill in match matrix  */ for (px = seqx[0], xx = 1; xx <=len0; px++, xx++) { /* initialize first entry in col  */ if (endgaps) {if (xx == 1) col1[0] = delx = −(ins0+ins1); else col1[0] = delx =col0[0]−ins1; ndelx = xx; } else { col1[0] = 0; delx = −ins0; ndelx = 0;} ...nw for (py = seqx[1], yy = 1; yy <= len1; py++, yy++) { mis =col0[yy−1]; if (dna) mis + = (xbm[*px−‘A’]&xbm[*py−‘A’])? DMAT : DMIS;else mis += _day[*px−‘A’][*py−‘A’]; /* update penalty for del in x seq; * favor new del over ongong del  * ignore MAXGAP if weighting endgaps */ if (endgaps || ndely[yy] < MAXGAP) { if (col0[yy] − ins0 >=dely[yy]) { dely[yy] = col0[yy] − (ins0+ins1); ndely[yy] = 1; } else {dely[yy] −= ins1; ndely[yy]++; } } else { if (col0[yy] − (ins0+ins1) >=dely[yy]) { dely[yy] = col0[yy] − (ins0+ins1); ndely[yy] = 1; } elsendely[yy]++; } /* update penalty for del in y seq;  * favor new del overongong del  */ if (endgaps || ndelx < MAXGAP) { if(col1[yy−1] − ins0 >=delx) { delx = col1[yy−1] − (ins0+ins1); ndelx = 1; } else { delx −=ins1; ndelx++; } } else { if (col1[yy−1] − (ins0+ins1) > = delx) { delx= col1[yy−1] − (ins0+ins1); ndelx = 1; } else ndelx+ +; } /* pick themaximum score; we're favoring  * mis over any del and delx over dely  */...nw id = xx − yy + len1 − 1; if (mis >= delx && mis >= dely[yy])col1[yy] = mis; else if (delx > = dely[yy]) { col1[yy] = delx; ij =dx[id].ijmp; if (dx[id].jp.n[0] && (!dna || (ndelx > = MAXJMP && xx >dx[id].jp.x[ij]+MX) || mis > dx[id].score+DINS0)) { dx[id].ijmp+ +; if(++ij >= MAXJMP) { writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset =offset; offset += sizeof(struct jmp) + sizeof(offset); } }dx[id].jp.n[ij] = ndelx; dx[id].jp.x[ij] = xx; dx[id].score = delx; }else { col1[yy] = dely[yy]; ij = dx[id].ijmp; if (dx[id].jp.n[0] &&(!dna || (ndely[yy] > = MAXJMP && xx > dx[id].jp.x[ij]+MX) || mis >dx[id].score+DINS0)) { dx[id].ijmp++; if (++ij >= MAXJMP) {writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset = offset; offset +=sizeof(struct jmp) + sizeof(offset); } } dx[id].jp.n[ij] =−ndely[yy];dx[id].jp.x[ij] = xx; dx[id].score = dely[yy]; } if (xx == len0 && yy <len1) { /* last col  */ if (endgaps) col1[yy] −= ins0+ins1*(len1−yy);if(col1[yy] > smax) { smax = col1[yy]; dmax = id; } } } if (endgaps &&xx < len0) col1[yy−1] −= ins0+ins1*(len0−xx); if (col1[yy−1] > smax) {smax = col1[yy−1]; dmax = id; } tmp = col0; col0 = col1; col1 = tmp; }(void) free((char *)ndely); (void) free((char *)dely); (void) free((char*)col0); (void) free((char *)col1); } /*  *  * print() -- only routinevisible outside this module  *  * static:  * getmat() -- trace back bestpath, count matches: print()  * pr_align() -- print alignment ofdescribed in array p[]: print()  * dumpblock() -- dump a block of lineswith numbers, stars: pr_align()  * nums() -- put out a number line:dumpblock()  * putline() -- put out a line (name, [num], seq, [num]):dumpblock()  * stars() - -put a line of stars: dumpblock()  *stripname() -- strip any path and prefix from a seqname  */ #include“nw.h” #define SPC  3 #define P_LINE 256 /* maximum output line */#define P_SPC  3 /* space between name or num and seq */ extern_day[26][26]; int olen; /* set output line length */ FILE *fx; /* outputfile */ print() print { int lx, ly, firstgap, lastgap;  /* overlap */ if((fx = fopen(ofile, “w”)) == 0) { fprintf(stderr, “ %s: can't write%s\n”, prog, ofile); cleanup(1); } fprintf(fx, “<first sequence: %s(length = %d)\n”, namex[0], len0); fprintf(fx, “<second sequence: %s(length = %d)\n”, namex[1], len1); olen = 60; lx = len0; ly = len1;firstgap = lastgap = 0; if (dmax < len1 − 1) { /* leading gap in x */pp[0].spc = firstgap = len1 − dmax − 1; ly −= pp[0].spc; } else if(dmax > len1 − 1) { /* leading gap in y */ pp[1].spc = firstgap = dmax −(len1 − 1); lx −= pp[1].spc; } if (dmax0 < len0 − 1) { /* trailing gapin x */ lastgap = len0 − dmax0 −1; lx −= lastgap; } else if (dmax0 >len0 − 1) { /* trailing gap in y */ lastgap = dmax0 − (len0 − 1); ly −=lastgap; } getmat(lx, ly, firstgap, lastgap); pr_align(); } /*  * traceback the best path, count matches  */ static getmat(lx, ly, firstgap,lastgap) getmat int lx, ly; /* “core” (minus endgaps) */ int firstgap,lastgap; /* leading trailing overlap */ { int nm, i0, i1, siz0, siz1;char outx[32]; double pct; register n0, n1; register char *p0, *p1; /*get total matches, score  */ i0 = i1 = siz0 = siz1 = 0; p0 = seqx[0] +pp[1].spc; p1 = seqx[1] + pp[0].spc; n0 = pp[1].spc + 1; n1 =pp[0].spc + 1; nm = 0; while ( *p0 && *p1 ) { if (siz0) { p1++; n1++;siz0−−; } else if (siz1) { p0+ +; n0+ +; siz1−−; } else { if(xbm[*p0−‘A’]&xbm[*p1−‘A’]) nm+ +; if (n0++ == pp[0].x[i0]) siz0 =pp[0].n[i0++]; if (nl++ == pp[1].x[i1]) siz1 = pp[1].n[il++]; p0+ +;p1++; } } /* pct homology:  * if penalizing endgaps, base is the shorterseq  * else, knock off overhangs and take shorter core  */ if (endgaps)lx = (len0 < len1)? len0 : len1; else lx = (lx < ly)? lx : ly; pct =100.*(double)nm/(double)lx; fprintf(fx, “\n”); fprintf(fx, “< %d match%sin an overlap of %d: %.2f percent similarity\n”, nm, (nm == 1)? “” :“es”, lx, pct); fprintf(fx, “<gaps in first sequence: %d”, gapx);...getmat if (gapx) { (void) sprintf(outx, “ (%d %s%s)”, ngapx, (dna)?“base”: “residue”, (ngapx == 1)? “”:“s”); fprintf(fx, “% s”, outx);fprintf(fx, “, gaps in second sequence: %d”, gapy); if (gapy) { (void)sprintf(outx, “(%d %s%s)”, ngapy, (dna)? “base”:“residue”, (ngapy == 1)?“”:“s”); fprintf(fx, “%s”, outx); } if (dna) fprintf(fx, “\n<score: %d(match = %d, mismatch = %d, gap penalty = %d + %d per base)\n”, smax,DMAT, DMIS, DINS0, DINS1); else fprintf(fx, “\n< score: %d (Dayhoff PAM250 matrix, gap penalty = %d + %d per residue)\n”, smax, PINS0, PINS1);if (endgaps) fprintf(fx, “<endgaps penalized. left endgap: %d %s%s,right endgap: %d %s%s\n”, firstgap, (dna)? “base” : “residue”, (firstgap== 1)? “” : “s”, lastgap, (dna)? “base” : “residue”, (lastgap == 1)? “”: “s”); else fprintf(fx, “<endgaps not penalized\n”); } static nm; /*matches in core -- for checking */ static lmax; /* lengths of strippedfile names */ static ij[2]; /* jmp index for a path */ static nc[2]; /*number at start of current line */ static ni[2]; /* current elem number-- for gapping */ static siz[2]; static char *ps[2]; /* ptr to currentelement */ static char *po[2]; /* ptr to next output char slot */ staticchar out[2][P_LINE]; /* output line */ static char star[P_LINE]; /* setby stars() */ /*  * print alignment of described in struct path pp[]  */static pr_align() pr_align { int nn; /* char count */ int more; registeri; for (i = 0, lmax = 0; i < 2; i++) { nn = stripname(namex[i]); if(nn > lmax) lmax = nn; nc[i] = 1; ni[i] = 1; siz[i] = ij[i] = 0; ps[i] =seqx[i]; po[i] = out[i]; } for (nn = nm = 0, more = 1; more;) {...pr_align for (i = more = 0; i < 2; i++) { /*  * do we have more ofthis sequence?  */ if (!*ps[i]) continue; more++; if (pp[i].spc) { /*leading space */ *po[i]++ = ‘ ’; pp[i].spc−−; } else if (siz[i]) { /* ina gap */ *po[i]++ = ‘−’; siz[i]−−; } else { /* we're putting a seqelement */ *po[i] = *ps[i]; if (islower(*ps[i])) *ps[i] =toupper(*ps[i]); po[i]++; ps[i]++; /*  * are we at next gap for thisseq?  */ if (ni[i] == pp[i].x[ij[i]]) { /*  * we need to merge all gaps * at this location  */ siz[i] == pp[i].n[ij[i]++]; while (ni[i] ==pp[i].x[ij[i]]) siz[i] += pp[i].n[ij[i]++]; } ni[i]++; } } if (++nn ==olen || !more && nn) { dumpblock(); for (i = 0; i < 2; i++) po[i] =out[i]; nn = 0; } } } /*  * dump a block of lines, including numbers,stars: pr_align()  */ static dumpblock() dumpblock { register i; for(i =0; i < 2; i++) *po[i]−− = ‘\0’; ...dumpblock (void) putc(‘\n’, fx); for(i = 0; i < 2; i++) { if (*out[i] && (*out[i] != ‘ ’ || *(po[i]) != ‘’)) { if (i == 0) nums(i); if (i == 0 && *out[1]) stars(); putline(i);if (i == 0 && *out[1]) fprintf(fx, star); if (i == 1) nums(i); } } }/* * put out a number line: dumpblock()  */ static nums(ix) numsint  ix; /* index in out[] holding seq line */ { char nline[P_LINE];register i, j; register char *pn, *px, *py; for(pn = nline, i = 0; i <lmax+P_SPC; i++, pn++) *pn = ‘ ’; for (i = nc[ix], py = out[ix]; *py;py++, pn++) { if (*py == ‘ ’ || *py == ‘−’); *pn = ‘ ’; else { if (i%10== 0 || (i == 1 && nc[ix] != 1)) { j = (i < 0)? −i : i; for (px = pn; j;j/= 10, px−−) *px = j%10 + ‘0’; if (i < 0) *px = ‘−’; } else *pn = ‘ ’;i+ +; } } *pn = ‘\0’; nc[ix] = i; for (pn = nline; *pn; pn+ +) (void)putc(*pn, fx); (void) putc(‘\n’, fx); } /*  * put out a line (name,[num], seq. [num]): dumpblock()  */ static putline(ix) putline int   ix;{ ...putline int i; register char *px; for (px = namex[ix], i = 0; *px&& *px != ‘:’; px++, i++) (void) putc(*px, fx); for (;i < lmax+P_SPC;i++) (void) putc(‘ ’, fx); /* these count from 1:  * ni[] is currentelement (from 1)  * nc[] is number at start of current line  */ for (px= out[ix]; *px; px+ +) (void) putc(*px&0x7F, fx); (void) putc(‘\n’, fx);} /*  * put a line of stars (seqs always in out[0], out[1]): dumpblock() */ static stars() stars { int i; register char *p0, *p1, cx, *px; if(!*out[0] || (*out[0] == ‘ ’ && *(p0[0]) == ‘ ’) || !*out[1] || (*out[1]== ‘ ’ && *(po[1]) == ‘ ’)) return; px = star; for (i = lmax+P_ SPC; i;i−−) *px++ = ‘ ’; for (p0 = out[0], p1 = out[1]; *p0 && *p1; p0++, p1++){ if (isalpha(*p0) && isalpha(*p1)) { if (xbm[*p0−‘A’]&xbm[*p1−‘A’]) {cx = ‘*’; nm+ +; } else if (!dna && _day[*p0− ‘A’][*p1−‘A’] > 0) cx =‘.’; else cx = ‘ ’; } else cx = ‘ ’; *px++ = cx; } *px++ = ‘\n’; *px =‘\0’; } /*  * strip path or prefix from pn, return len: pr_align()  */static stripname(pn) stripname char *pn; /* file name (may be path) */ {register char *px, *py; py = 0; for (px = pn; *px; px++) if (*px == ‘/’)py = px + 1; if (py) (void) strcpy(pn, py); return(strlen(pn)); } /*  *cleanup() -- cleanup any tmp file  * getseq() -- read in seq, set dna,len, maxlen  * g_calloc() -- calloc() with error checkin  * readjmps()-- get the good jmps, from tmp file if necessary  * writejmps() -- writea filled array of jmps to a tmp file: nw()  */ #include “nw.h” #include<sys/file.h> char *jname = “/tmp/homgXXXXXX”; /* tmp file for jmps */FILE *fj; int cleanup(); /* cleanup tmp file */ long lseek(); /*  *remove any tmp file if we blow  */ cleanup(i) cleanup int i; { if (fj)(void) unlink(jname); exit(i); } /*  * read, return ptr to seq, set dna,len, maxlen  * skip lines starting with ‘;’, ‘<’, or ‘>’  * seq in upperor lower case  */ char * getseq(file, len) getseq char *file; /* filename */ int *len; /* seq len */ { char line[1024], *pseq; register char*px, *py; int natgc, tlen; FILE *fp; if ((fp = fopen(file, “r”)) == 0) {fprintf(stderr, “%s: can't read %s\n”, prog, file); exit(1); } tlen =natgc = 0; while (fgets(line, 1024, fp)) { if (*line == ‘;’ || *line ==‘<’ || *line == ‘>’) continue; for (px = line; *px != ‘\n’; px+ +) if(isupper(*px) || islower(*px)) tlen++; } if ((pseq =malloc((unsigned)(tlen+ 6))) == 0) { fprintf(stderr, “%s: malloc()failed to get %d bytes for %s\n”, prog, tlen+6, file); exit(1); }pseq[0] = pseq[1] = pseq[2] = pseq[3] = ‘\0’; ...getseq py = pseq + 4;*len = tlen; rewind(fp); while (fgets(line, 1024, fp)) { if (*line ==‘;’ || *line == ‘<’ || *line == ‘>’) continue; for (px = line; *px !=‘\n’; px++) { if (isupper(*px)) *py++ = *px; else if (islower(*px))*py+ + = toupper(*px); if (index(“ATGCU” , *(py−1))) natgc+ +; } } *py++= ‘\0’; *py = ‘\0’; (void) fclose(fp); dna = natgc > (tlen/3);return(pseq+4); } char * g_calloc(msg, nx, sz) g_calloc char *msg; /*program, calling routine */ int nx, sz; /* number and size of elements*/ { char *px, *calloc(); if ((px = calloc((unsigned)nx, (unsigned)sz))== 0) { if (*msg) { fprintf(stderr, “%s: g_calloc() failed %s (n= %d,sz= %d)\n”, prog, msg, nx, sz); exit(1); } } return(px); } /*  * getfinal jmps from dx[] or tmp file, set pp[], reset dmax: main()  */readjmps() readjmps { int fd = −1; int siz, i0, i1; register i, j, xx;if (fj) { (void) fclose(fj); if ((fd = open(jname, O_RDONLY, 0)) < 0) {fprintf(stderr, “%s: can't open() %s\n”, prog, jname); cleanup(1); } }for (i = i0 = i1 = 0, dmax0 = dmax, xx = len0; ;i++) { while (1) { for(j = dx[dmax].ijmp; j >= 0 && dx[dmax].jp.x[j] >= xx; j−−) ; ...readjmpsif (j < 0 && dx[dmax].offset && fj) { (void) lseek(fd, dx[dmax].offset,0); (void) read(fd, (char *)&dx[dmax].jp, sizeof(struct jmp)); (void)read(fd, (char *)&dx[dmax].offset, sizeof(dx[dmax].offset));dx[dmax].ijmp = MAXJMP−1; } else break; } if (i > = JMPS) {fprintf(stderr, “%s: too many gaps in alignment\n”, prog); cleanup(1); }if (j >= 0) { siz = dx[dmax].jp.n[j]; xx = dx[dmax].jp.x[j]; dmax +=siz; if (siz < 0) { /* gap in second seq */ pp[1].n[il] = −siz; xx +=siz; /* id = xx − yy + len1 − 1  */ pp[1].x[il] = xx − dmax + len1 − 1;gapy+ +; ngapy −= siz; /* ignore MAXGAP when doing endgaps */ siz =(−siz < MAXGAP || endgaps)? −siz : MAXGAP; il++; } else if (siz > 0) {/* gap in first seq */ pp[0] .n[i0] = siz; pp[0] .x[i0] = xx; gapx+ +;ngapx += siz; /* ignore MAXGAP when doing endgaps */ siz = (siz < MAXGAP|| endgaps)? siz : MAXGAP; i0+ +; } } else break; } /* reverse the orderof jmps  */ for (j = 0, i0−−; j < i0; j++, i0−−) { i = pp[0].n[j];pp[0].n[j] = pp[0].n[i0]; pp[0].n[i0] = i; i = pp[0].x[j]; pp[0].x[j] =pp[0].x[i0]; pp[0].x[i0] = i; } for (j = 0, i1−−; j < i1; j++, i1−−) { i= pp[1].n[j]; pp[1].n[j] = pp[1].n[i1]; pp[1].n[i1] = i; i = pp[1].x[j];pp[1].x[j] = pp[1].x[i1]; pp[1].x[i1] = i; } if (fd > = 0) (void)close(fd); if (fj) { (void) unlink(jname); fj = 0; offset = 0; } } /*  *write a filled jmp struct offset of the prev one (if any): nw()  */writejmps(ix) writejmps int ix; { char *mktemp(); if (!fj) { if(mktemp(jname) < 0) { fprintf(stderr, “%s: can't mktemp() %s\n”, prog,jname); cleanup(1); } if ((fj = fopen(jname, “w”)) == 0) {fprintf(stderr, “%s: can't write %s\n”, prog, jname); exit(1); } }(void) fwrite((char *)&dx[ix].jp, sizeof(struct jmp), 1, fj); (void)fwrite((char *)&dx[ix].offset, sizeof(dx[ix].offset), 1, fj); }

TABLE 2 PRO XXXXXXXXXXXXXXX (Length = 15 amino acids) ComparisonXXXXXYYYYYYY (Length = 12 amino acids) Protein % amino acid sequenceidentity = (the number of identically matching amino acid residuesbetween the two polypeptide sequences as determined by ALIGN-2) dividedby (the total number of amino acid residues of the PRO polypeptide) = 5divided by 15 = 33.3%

TABLE 3 PRO XXXXXXXXXX (Length = 10 amino acids) ComparisonXXXXXYYYYYYZZYZ (Length = 15 amino acids) Protein % amino acid sequenceidentity = (the number of identically matching amino acid residuesbetween the two polypeptide sequences as determined by ALIGN-2) dividedby (the total number of amino acid residues of the PRO polypeptide) = 5divided by 10 = 50%

TABLE 4 PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides) ComparisonNNNNNNLLLLLLLLLL (Length = 16 nucleotides) DNA % nucleic acid sequenceidentity = (the number of identically matching nucleotides between thetwo nucleic acid sequences as determined by ALIGN-2) divided by (thetotal number of nucleotides of the PRO-DNA nucleic acid sequence) = 6divided by 14 = 42.9%

TABLE 5 PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides) Comparison DNANNNNLLLVV (Length = 9 nucleotides) % nucleic acid sequence identity =(the number of identically matching nucleotides between the two nucleicacid sequences as determined by ALIGN-2) divided by (the total number ofnucleotides of the PRO-DNA nucleic acid sequence) = 4 divided by 12 =33.3%II. Compositions and Methods of the Invention

A. Full-length PRO Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO polypeptides. In particular, cDNAs encoding various PROpolypeptides have been identified and isolated, as disclosed in furtherdetail in the Examples below. It is noted that proteins produced inseparate expression rounds may be given different PRO numbers but theUNQ number is unique for any given DNA and the encoded protein, and willnot be changed. However, for sake of simplicity, in the presentspecification the protein encoded by the full length native nucleic acidmolecules disclosed herein as well as all further native homologues andvariants included in the foregoing definition of PRO, will be referredto as “PRO/number”, regardless of their origin or mode of preparation.

As disclosed in the Examples below, various cDNA clones have beendeposited with the ATCC. The actual nucleotide sequences of those clonescan readily be determined by the skilled artisan by sequencing of thedeposited clone using routine methods in the art. The predicted aminoacid sequence can be determined from the nucleotide sequence usingroutine skill. For the PRO polypeptides and encoding nucleic acidsdescribed herein, Applicants have identified what is believed to be thereading frame best identifiable with the sequence information availableat the time.

1. Full-length PRO211 and PRO217 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO211 and PRO217. In particular, Applicants have identified andisolated cDNA encoding PRO211 and PRO217 polypeptides, as disclosed infurther detail in the Examples below. Using BLAST (FastA format)sequence alignment computer programs, Applicants found that cDNAsequences encoding full-length native sequence PRO211 and PRO217 havehomologies to known proteins having EGF-like domains. Specifically, thecDNA sequence DNA32292-1131 (FIG. 1, SEQ ID NO:1) has certain identifyand a Blast score of 209 with PAC6_RAT and certain identify and a Blastscore of 206 with Fibulin-1, isoform c precursor. The cDNA sequenceDNA33094-1131 (FIG. 3, SEQ ID NO:3) has 36% identity and a Blast scoreof 336 with eastern newt tenascin, and 37% identity and a Blast score of331 with human tenascin-X precursor. Accordingly, it is presentlybelieved that PRO211 and PRO217 polypeptides disclosed in the presentapplication are newly identified members of the EGF-like family andpossesses properties typical of the EGF-like protein family.

2. Full-length PRO230 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO230. In particular, Applicants have identified and isolated cDNAencoding a PRO230 polypeptide, as disclosed in further detail in theExamples below. Using known programs such as BLAST and FastA sequencealignment computer programs, Applicants found that a cDNA sequenceencoding full-length native sequence PRO230 has 48% amino acid identitywith the rabbit tubulointerstitial nephritis antigen precursor.Accordingly, it is presently believed that PRO230 polypeptide disclosedin the present application is a newly identified member of thetubulointerstitial nephritis antigen family and possesses the ability tobe recognized by human autoantibodies in certain forms oftubulointerstitial nephritis.

-   -   3. Full-length PRO232 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO232. In particular, Applicants have identified and isolated cDNAencoding a PRO232 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that a portion of the full-length nativesequence PRO232 (shown in FIG. 9 and SEQ ID NO:18) has 35% sequenceidentity with a stem cell surface antigen from Gallus gallus.Accordingly, it is presently believed that the PRO232 polypeptidedisclosed in the present application may be a newly identified stem cellantigen.

-   -   4. Full-length PRO187 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO187. In particular, Applicants have identified and isolated cDNAencoding a PRO187 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that a full-length native sequence PRO187(shown in FIG. 15) has 74% amino acid sequence identity and BLAST scoreof 310 with various androgen-induced growth factors and FGF-8.Accordingly, it is presently believed that PRO187 polypeptide disclosedin the present application is a newly identified member of the FGF-8protein family and may possess identify activity or property typical ofthe FGF-8-like protein family.

-   -   5. Full-length PRO265 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO265. In particular, Applicants have identified and isolated cDNAencoding a PRO265 polypeptide, as disclosed in further detail in theExamples below. Using programs such as BLAST and FastA sequencealignment computer programs, Applicants found that various portions ofthe PRO265 polypeptide have significant homology with the fibromodulinprotein and fibromodulin precursor protein. Applicants have also foundthat the DNA encoding the PRO265 polypeptide has significant homologywith platelet glycoprotein V, a member of the leucine rich relatedprotein family involved in skin and wound repair. Accordingly, it ispresently believed that PRO265 polypeptide disclosed in the presentapplication is a newly identified member of the leucine rich repeatfamily and possesses protein protein binding capabilities, as well as beinvolved in skin and wound repair as typical of this family.

-   -   6. Full-length PRO219 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO219. In particular, Applicants have identified and isolated cDNAencoding a PRO219 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO219polypeptide have significant homology with the mouse and humanmatrilin-2 precursor polypeptides. Accordingly, it is presently believedthat PRO219 polypeptide disclosed in the present application is relatedto the matrilin-2 precursor polypeptide.

-   -   7. Full-length PRO246 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO246. In particular, Applicants have identified and isolated cDNAencoding a PRO246 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that a portion of the PRO246 polypeptide hassignificant homology with the human cell surface protein HCAR.Accordingly, it is presently believed that PRO246 polypeptide disclosedin the present application may be a newly identified membrane-boundvirus receptor or tumor cell-specific antigen.

-   -   8. Full-length PRO228 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO228. In particular, Applicants have identified and isolated cDNAencoding a PRO228 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO228polypeptide have significant homology with the EMR1 protein. Applicantshave also found that the DNA encoding the PRO228 polypeptide hassignificant homology with latrophilin, macrophage-restricted cellsurface glycoprotein, B0457.1 and leucocyte antigen CD97 precursor.Accordingly, it is presently believed that PRO228 polypeptide disclosedin the present application is a newly identified member of the seventransmembrane superfamily and possesses characteristics and functionalproperties typical of this family. In particular, it is believed thatPRO228 is a new member of the subgroup within this family to which CD97and EMR1 belong.

-   -   9. Full-length PRO533 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO533. In particular, Applicants have identified and isolated cDNAencoding a PRO533 polypeptide, as disclosed in further detail in theExamples below. Using BLAST-2 and FastA sequence alignment computerprograms, Applicants found that a full-length native sequence PRO533(shown in FIG. 22 and SEQ ID NO:59) has a Blast score of 509 and 53%amino acid sequence identity with fibroblast growth factor (FGF).Accordingly, it is presently believed that PRO533 disclosed in thepresent application is a newly identified member of the fibroblastgrowth factor family and may possess activity typical of suchpolypeptides.

-   -   10. Full-length PRO245 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO245. In particular, Applicants have identified and isolated cDNAencoding a PRO245 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that a portion of the amino acid sequence ofthe PRO245 polypeptide has 60% amino acid identity with the human c-mybprotein. Accordingly, it is presently believed that the PRO245polypeptide disclosed in the present application may be a newlyidentified member of the transmembrane protein tyrosine kinase family.

-   -   11. Full-length PRO220, PRO221 and PRO227 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO220, PRO221 and PRO227. In particular, Applicants have identifiedand isolated cDNAs encoding a PRO220, PRO221 and PRO227 polypeptide,respectively, as disclosed in further detail in the Examples below.Using BLAST and FastA sequence alignment computer programs, PRO220 hasamino acid identity with the amino acid sequence of a leucine richprotein wherein the identity is 87%. PRO220 additionally has amino acididentity with the neuronal leucine rich protein wherein the identity is55%. The neuronal leucine rich protein is further described in Taguchi,et al., Mol. Brain Res., 35:31-40 (1996).

PRO221 has amino acid identity with the SLIT protein precursor, whereindifferent portions of these two proteins have the respective percentidentities of 39%, 38%, 34%, 31%, and 30%.

PRO227 has amino acid identity with the amino acid sequence of plateletglycoprotein V precursor. The same results were obtained for humanglycoprotein V. Different portions of these two proteins show thefollowing percent identities of 30%, 28%, 28%, 31%, 35%, 39% and 27%.

Accordingly, it is presently believed that PRO220, PRO221 and PRO227polypeptides disclosed in the present application are newly identifiedmembers of the leucine rich repeat protein superfamily and that eachpossesses protein-protein binding capabilities typical of the leucinerich repeat protein superfamily. It is also believed that they havecapabilities similar to those of SLIT, the leucine rich repeat proteinand human glycoprotein V.

-   -   12. Full-length PRO258 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO258. In particular, Applicants have identified and isolated cDNAencoding a PRO258 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO258polypeptide have significant homology with the CRTAM and poliovirusreceptors. Accordingly, it is presently believed that PRO258 polypeptidedisclosed in the present application is a newly identified member of theIg superfamily and possesses virus receptor capabilities or regulatesimmune function as typical of this family.

-   -   13. Full-length PRO266 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO266. In particular, Applicants have identified and isolated cDNAencoding a PRO266 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO266polypeptide have significant homology with the SLIT protein fromDrosophilia. Accordingly, it is presently believed that PRO266polypeptide disclosed in the present application is a newly identifiedmember of the leucine rich repeat family and possesses ligand-ligandbinding activity and neuronal development typical of this family. SLIThas been shown to be useful in the study and treatment of Alzheimer'sdisease, supra, and thus, PRO266 may have involvement in the study andcure of this disease.

-   -   14. Full-length PRO269 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO269. In particular, Applicants have identified and isolated cDNAencoding a PRO269 polypeptide, as disclosed in further detail in theExamples below. Using BLAST, FastA and sequence alignment computerprograms, Applicants found that the amino acid sequence encoded bynucleotides 314 to 1783 of the full-length native sequence PRO269 (shownin FIG. 35 and SEQ ID NO:95) has significant homology to human urinarythrombomodulin and various thrombomodulin analogues respectively, towhich it was aligned. Accordingly, it is presently believed that PRO269polypeptide disclosed in the present application is a newly identifiedmember of the thrombomodulin family.

-   -   15. Full-length PRO287 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO287. In particular, Applicants have identified and isolated cDNAencoding a PRO287 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO287polypeptide have significant homology with the type 1 procollagenC-proteinase enhancer protein precursor and type 1 procollagenC-proteinase enhancer protein. Accordingly, it is presently believedthat PRO287 polypeptide disclosed in the present application is a newlyidentified member of the C-proteinase enhancer protein family.

-   -   16. Full-length PRO214 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO214. In particular, Applicants have identified and isolated cDNAencoding a PRO214 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that a full-length native sequence PRO214polypeptide (shown in FIG. 40 and SEQ ID NO:109) has 49% amino acidsequence identity with HT protein, a known member of the EGF-family. Thecomparison resulted in a BLAST score of 920, with 150 matchingnucleotides. Accordingly, it is presently believed that the PRO214polypeptide disclosed in the present application is a newly identifiedmember of the family comprising EGF domains and may possess activitiesor properties typical of the EGF-domain containing family.

-   -   17. Full-length PRO317 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO317. In particular, cDNA encoding a PRO317 polypeptide has beenidentified and isolated, as disclosed in further detail in the Examplesbelow. Using BLAST™ and FastA™ sequence alignment computer programs, itwas found that a full-length native-sequence PRO317 (shown in FIG. 42and SEQ ID NO:114) has 92% amino acid sequence identity with EBAF-1.Further, it is closely aligned with many other members of theTGF-superfamily.

Accordingly, it is presently believed that PRO317 disclosed in thepresent application is a newly identified member of the TGF-superfamilyand may possess properties that are therapeutically useful in conditionsof uterine bleeding, etc. Hence, PRO317 may be useful in diagnosing ortreating abnormal bleeding involved in gynecological diseases, forexample, to avoid or lessen the need for a hysterectomy. PRO317 may alsobe useful as an agent that affects angiogenesis in general, so PRO317may be useful in anti-tumor indications, or conversely, in treatingcoronary ischemic conditions.

Library sources reveal that ESTs used to obtain the consensus DNA forgenerating PRO317 primers and probes were found in normal tissues(uterus, prostate, colon, and pancreas), in several tumors (colon, brain(twice), pancreas, and mullerian cell), and in a heart with ischemia.PRO317 has shown up in several tissues as well, but it does look to havea greater concentration in uterus. Hence, PRO317 may have a broader useby the body than EBAF-1. It is contemplated that, at least for someindications, PRO317 may have opposite effects from EBAF-1.

-   -   18. Full-length PRO301 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO301. In particular, Applicants have identified and isolated cDNAencoding a PRO301 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that a full-length native sequence PRO301(shown in FIG. 44 and SEQ ID NO:119) has a Blast score of 246corresponding to 30% amino acid sequence identity with human A33 antigenprecursor. Accordingly, it is presently believed that PRO301 disclosedin the present application is a newly identified member of the A33antigen protein family and may be expressed in human neoplastic diseasessuch as colorectal cancer.

-   -   19. Full-length PRO224 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO224. In particular, Applicants have identified and isolated cDNAencoding a PRO224 polypeptide, as disclosed in further detail in theExamples below. Using known programs such as BLAST and FastA sequencealignment computer programs, Applicants found that full-length nativePRO224 (FIG. 46, SEQ ID NO:127) has amino acid identity withapolipoprotein E receptor 2906 from homo sapiens. The alignments ofdifferent portions of these two polypeptides show amino acid identitiesof 37%, 36%, 30%, 44%, 44% and 28% respectively. Full-length nativePRO224 (FIG. 46, SEQ ID NO:127) also has amino acid identity with verylow-density lipoprotein receptor precursor from gall. The alignments ofdifferent portions of these two polypeptides show amino acid identitiesof 38%, 37%, 42%, 33%, and 37% respectively. Additionally, full-lengthnative PRO224 (FIG. 46, SEQ ID NO:127) has amino acid identity with thechicken oocyte receptor P95 from Gallus gallus. The alignments ofdifferent portions of these two polypeptides show amino acid identitiesof 38%, 37%, 42%, 33%, and 37% respectively. Moreover, full-lengthnative PRO224 (FIG. 46, SEQ ID NO:127) has amino acid identity with verylow density lipoprotein receptor short form precursor from humans. Thealignments of different portions of these two polypeptides show aminoacid identities of 32%, 38%, 34%, 45%, and 31%, respectively.Accordingly, it is presently believed that PRO224 polypeptide disclosedin the present application is a newly identified member of the lowdensity lipoprotein receptor family and possesses the structuralcharacteristics required to have the functional ability to recognize andendocytose low density lipoproteins typical of the low densitylipoprotein receptor family. (The alignments described above used thefollowing scoring parameters: T=7, S+65, S2=36, Matrix: BLOSUM62.)

-   -   20. Full-length PRO222 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO222. In particular, Applicants have identified and isolated cDNAencoding a PRO222 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that a sequence encoding full-length nativesequence PRO222 (shown in FIG. 48 and SEQ ID NO:132) has 25-26% aminoacid identity with mouse complement factor h precursor, has 27-29% aminoacid identity with complement receptor, has 25-47% amino acid identitywith mouse complement C3b receptor type 2 long form precursor, has 40%amino acid identity with human hypothetical protein kiaa0247.Accordingly, it is presently believed that PRO222 polypeptide disclosedin the present application is a newly identified member of thecomplement receptor family and possesses activity typical of thecomplement receptor family.

-   -   21. Full-length PRO234 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO234. In particular, Applicants have identified and isolated cDNAencoding a PRO234 polypeptide, as disclosed in further detail in theExamples below. Using BLAST (FastA-format) sequence alignment computerprograms, Applicants found that a cDNA sequence encoding full-lengthnative sequence PRO234 has 31% identity and Blast score of 134 withE-selectin precursor. Accordingly, it is presently believed that thePRO234 polypeptides disclosed in the present application are newlyidentified members of the lectin/selectin family and possess activitytypical of the lectin/selectin family.

-   -   22. Full-length PRO231 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO231. In particular, Applicants have identified and isolated cDNAencoding a PRO231 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the full-length native sequence PRO231polypeptide (shown in FIG. 52 and SEQ ID NO:142) has 30% and 31% aminoacid identity with human and rat prostatic acid phosphatase precursorproteins, respectively. Accordingly, it is presently believed that thePRO231 polypeptide disclosed in the present application may be a newlyidentified member of the acid phosphatase protein family.

-   -   23. Full-length PRO229 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO229. In particular, Applicants have identified and isolated cDNAencoding a PRO229 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO229polypeptide have significant homology with antigen wc1.1, M130 antigen,T cell surface glycoprotein CD6 and CD6. It also is related to Sp-alpha.Accordingly, it is presently believed that PRO229 polypeptide disclosedin the present application is a newly identified member of the familycontaining scavenger receptor homology, a sequence motif found in anumber of proteins involved in immune function and thus possesses immunefunction and/or segments which resist degradation, typical of thisfamily.

-   -   24. Full-length PRO238 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO238. In particular, Applicants have identified and isolated cDNAencoding a PRO238 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO238polypeptide have significant homology with reductases, includingoxidoreductase and fatty acyl-CoA reductase. Accordingly, it ispresently believed that PRO238 polypeptide disclosed in the presentapplication is a newly identified member of the reductase family andpossesses reducing activity typical of the reductase family.

-   -   25. Full-length PRO233 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO233. In particular, Applicants have identified and isolated cDNAencoding a PRO233 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO233polypeptide have significant homology with the reductase protein.Applicants have also found that the DNA encoding the PRO233 polypeptidehas significant homology with proteins from Caenorhabditis elegans.Accordingly, it is presently believed that PRO233 polypeptide disclosedin the present application is a newly identified member of the reductasefamily and possesses the ability to effect the redox state of the celltypical of the reductase family.

-   -   26. Full-length PRO223 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO223. In particular, Applicants have identified and isolated cDNAencoding a PRO223 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO223 polypeptide has significanthomology with various serine carboxypeptidase polypeptides. Accordingly,it is presently believed that PRO223 polypeptide disclosed in thepresent application is a newly identified serine carboxypeptidase.

-   -   27. Full-length PRO235 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO235. In particular, Applicants have identified and isolated cDNAencoding a PRO235 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO235polypeptide have significant homology with the various plexin proteins.Accordingly, it is presently believed that PRO235 polypeptide disclosedin the present application is a newly identified member of the plexinfamily and possesses cell adhesion properties typical of the plexinfamily.

-   -   28. Full-length PRO236 and PRO262 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO236 and PRO262. In particular, Applicants have identified andisolated cDNA encoding PRO236 and PRO262 polypeptides, as disclosed infurther detail in the Examples below. Using BLAST and FastA sequencealignment computer programs, Applicants found that various portions ofthe PRO236 and PRO262 polypeptides have significant homology withvarious β-galactosidase and β-galactosidase precursor polypeptides.Accordingly, it is presently believed that the PRO236 and PRO262polypeptides disclosed in the present application are newly identifiedβ-galactosidase homologs.

-   -   29. Full-length PRO239 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO239. In particular, Applicants have identified and isolated cDNAencoding a PRO239 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO239polypeptide have significant homology with densin proteins. Accordingly,it is presently believed that PRO239 polypeptide disclosed in thepresent application is a newly identified member of the densin familyand possesses cell adhesion and the ability to effect synaptic processesas is typical of the densin family.

-   -   30. Full-length PRO257 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO257. In particular, Applicants have identified and isolated cDNAencoding a PRO257 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO257polypeptide have significant homology with the ebnerin precursor andebnerin protein. Accordingly, it is presently believed that PRO257polypeptide disclosed in the present application is a newly identifiedprotein member which is related to the ebnerin protein.

-   -   31. Full-length PRO260 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO260. In particular, Applicants have identified and isolated cDNAencoding a PRO260 polypeptide, as disclosed in further detail in theExamples below. Using programs such as BLAST and FastA sequencealignment computer programs, Applicants found that various portions ofthe PRO260 polypeptide have significant homology with thealpha-1-fucosidase precursor. Accordingly, it is presently believed thatPRO260 polypeptide disclosed in the present application is a newlyidentified member of the fucosidase family and possesses enzymaticactivity related to fucose residues typical of the fucosidase family.

-   -   32. Full-length PRO263 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO263. In particular, Applicants have identified and isolated cDNAencoding a PRO263 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO263polypeptide have significant homology with the CD44 antigen and relatedproteins. Accordingly, it is presently believed that PRO263 polypeptidedisclosed in the present application is a newly identified member of theCD44 antigen family and possesses at least one of the propertiesassociated with these antigens, i.e., cancer and HIV marker, cell-cellor cell-matrix interactions, regulating cell traffic, lymph node homing,transmission of growth signals, and presentation of chemokines andgrowth facors to traveling cells.

-   -   33. Full-length PRO270 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO270. In particular, Applicants have identified and isolated cDNAencoding a PRO270 polypeptide, as disclosed in further detail in theExamples below. Using BLAST, FastA and sequence alignment computerprograms, Applicants found that that various portions of the PRO270polypeptide have significant homology with various thioredoxin proteins.Accordingly, it is presently believed that PRO270 polypeptide disclosedin the present application is a newly identified member of thethioredoxin family and possesses the ability to effectreduction-oxidation (redox) state typical of the thioredoxin family.

-   -   34. Full-length PRO271 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO271. In particular, Applicants have identified and isolated cDNAencoding a PRO271 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that the PRO271 polypeptide has significanthomology with various link proteins and precursors thereof. Accordingly,it is presently believed that PRO271 polypeptide disclosed in thepresent application is a newly identified link protein homolog.

-   -   35. Full-length PRO272 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO272. In particular, Applicants have identified and isolated cDNAencoding a PRO272 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO272polypeptide have significant homology with the human reticulocalbinprotein and its precursors. Applicants have also found that the DNAencoding the PRO272 polypeptide has significant homology with the mousereticulocalbin precursor protein. Accordingly, it is presently believedthat PRO272 polypeptide disclosed in the present application is a newlyidentified member of the reticulocalbin family and possesses the abilityto bind calcium typical of the reticulocalbin family.

-   -   36. Full-length PRO294 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO294. In particular, Applicants have identified and isolated cDNAencoding a PRO294 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO294polypeptide have significant homology with the various portions of anumber of collagen proteins. Accordingly, it is presently believed thatPRO294 polypeptide disclosed in the present application is a newlyidentified member of the collagen family.

-   -   37. Full-length PRO295 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO295. In particular, Applicants have identified and isolated cDNAencoding a PRO295 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO295polypeptide have significant homology with integrin proteins.Accordingly, it is presently believed that PRO295 polypeptide disclosedin the present application is a newly identified member of the integrinfamily and possesses cell adhesion typical of the integrin family.

-   -   38. Full-length PRO293 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO293. In particular, Applicants have identified and isolated cDNAencoding a PRO293 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that portions of the PRO293 polypeptide havesignificant homology with the neuronal leucine rich repeat proteins 1and 2, (NLRR-1 and NLRR-2), particularly NLRR-2. Accordingly, it ispresently believed that PRO293 polypeptide disclosed in the presentapplication is a newly identified member of the neuronal leucine richrepeat protein family and possesses ligand-ligand binding activitytypical of the NRLL protein family.

-   -   39. Full-length PRO247 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO247. In particular, Applicants have identified and isolated cDNAencoding a PRO247 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO247polypeptide have significant homology with densin. Applicants have alsofound that the DNA encoding the PRO247 polypeptide has significanthomology with a number of other proteins, including KIAA0231.Accordingly, it is presently believed that PRO247 polypeptide disclosedin the present application is a newly identified member of the leucinerich repeat family and possesses ligand binding abilities typical ofthis family.

-   -   40. Full-length PRO302, PRO303, PRO304, PRO307 and PRO343        Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO302, PRO303, PRO304, PRO307 and PRO343. In particular, Applicantshave identified and isolated cDNA encoding PRO302, PRO303, PRO304,PRO307 and PRO343 polypeptides, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO302, PRO303,PRO304, PRO307 and PRO343 polypeptides have significant homology withvarious protease proteins. Accordingly, it is presently believed thatthe PRO302, PRO303, PRO304, PRO307 and PRO343 polypeptides disclosed inthe present application are newly identified protease proteins.

-   -   41. Full-length PRO328 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO328. In particular, Applicants have identified and isolated cDNAencoding a PRO328 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO328polypeptide have significant homology with the human glioblastomaprotein (“GLIP”). Further, Applicants found that various portions of thePRO328 polypeptide have significant homology with the cysteine richsecretory protein (“CRISP”) as identified by BLAST homology[ECCRISP3_(—)1, S68683, and CRS3_HUMAN]. Accordingly, it is presentlybelieved that PRO328 polypeptide disclosed in the present application isa newly identified member of the GLIP or CRISP families and possessestranscriptional regulatory activity typical of the GLIP or CRISPfamilies.

-   -   42. Full-length PRO335, PRO331 and PRO326 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO335, PRO331 or PRO326. In particular, Applicants have identifiedand isolated cDNA encoding a PRO335, PRO331 or PRO326 polypeptide, asdisclosed in further detail in the Examples below. Using BLAST and FastAsequence alignment computer programs, Applicants found that variousportions of the PRO335, PRO331 or PRO326 polypeptide have significanthomology with LIG-1, ALS and in the case of PRO331, additionally,decorin. Accordingly, it is presently believed that the PRO335, PRO331and PRO326 polypeptides disclosed in the present application are newlyidentified members of the leucine rich repeat superfamily, andparticularly, are related to LIG-1 and possess the biological functionsof this family as discussed and referenced herein.

-   -   43. Full-length PRO332 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO332. In particular, Applicants have identified and isolated cDNAencoding PRO332 polypeptides, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that a full-length native sequence PRO332(shown in FIG. 108 and SEQ ID NO:310) has about 30-40% amino acidsequence identity with a series of known proteoglycan sequences,including, for example, fibromodulin and fibromodulin precursorsequences of various species (FMOD_BOVIN, FMOD_CHICK, FMOD_RAT,FMOD_MOUSE, FMOD_HUMAN, P_R36773), osteomodulin sequences(AB000114_(—)1, AB007848_(—)1), decorin sequences (CFU83141_(—)1,OCU03394_(—)1, P R42266, P_R42267, P_R42260, P R89439), keratan sulfateproteoglycans (BTU48360_(—)1, AF022890_(—)1), corneal proteoglycan(AF022256_(—)1), and bonelcartilage proteoglycans and proteoglycaneprecursors (PGS1_BOVIN, PGS2_MOUSE, PGS2_HUMAN). Accordingly, it ispresently believed that PRO332 disclosed in the present application is anew proteoglycan-type molecule, and may play a role in regulatingextracellular matrix, cartilage, and/or bone function.

-   -   44. Full-length PRO334 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO334. In particular, Applicants have identified and isolated cDNAencoding a PRO334 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO334polypeptide have significant homology with fibulin and fibrillin.Accordingly, it is presently believed that PRO334 polypeptide disclosedin the present application is a newly identified member of the epidermalgrowth factor family and possesses properties and activities typical ofthis family.

-   -   45. Full-length PRO346 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO346. In particular, Applicants have identified and isolated cDNAencoding a PRO346 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that a full-length native sequence PRO346(shown in FIG. 112 and SEQ ID NO:320) has 28% amino acid sequenceidentity with carcinoembryonic antigen. Accordingly, it is presentlybelieved that PRO346 disclosed in the present application is a newlyidentified member of the carcinoembryonic protein family and may beexpressed in association with neoplastic tissue disorders.

-   -   46. Full-length PRO268 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO268. In particular, Applicants have identified and isolated cDNAencoding a PRO268 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that portions of the PRO268 polypeptide havesignificant homology with the various protein disulfide isomeraseproteins. Accordingly, it is presently believed that PRO268 polypeptidedisclosed in the present application is a homolog of the proteindisulfide isomerase p5 protein.

-   -   47. Full-length PRO330 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO330. In particular, Applicants have identified and isolated cDNAencoding a PRO330 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO330polypeptide have significant homology with the murine prolyl4-hydroxylase alpha-II subunit protein. Accordingly, it is presentlybelieved that PRO330 polypeptide disclosed in the present application isa novel prolyl 4-hydroxylase subunit polypeptide.

-   -   48. Full-length PRO339 and PRO310 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO339 and PRO310. In particular, Applicants have identified andisolated cDNA encoding a PRO339 polypeptide, as disclosed in furtherdetail in the Examples below. Applicants have also identified andisolated cDNA encoding a PRO310 polypeptide, as disclosed in furtherdetail in the Examples below. Using BLAST and FastA sequence alignmentcomputer programs, Applicants found that various portions of the PRO339and PRO310 polypeptides have significant homology with small secretedproteins from C. elegans and are distantly related to fringe. PRO339also shows homology to collagen-like polymers. Sequences which were usedto identify PRO310, designated herein as DNA40533 and DNA42267, alsoshow homology to proteins from C. elegans. Accordingly, it is presentlybelieved that the PRO339 and PRO310 polypeptides disclosed in thepresent application are newly identified member of the family ofproteins involved in development, and which may have regulatoryabilities similar to the capability of fringe to regulate serrate.

-   -   49. Full-length PRO244 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding C-type lectins referred to in the present applicationas PRO244. In particular, applicants have identified and isolated cDNAencoding PRO244 polypeptides, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that a full-length native sequence PRO244(shown in FIG. 122 and SEQ ID NO:377) has 43% amino acid sequenceidentity with the hepatic lectin gallus gallus (LECH-CHICK), and 42%amino acid sequence identity with an HIV gp120 binding C-type lectin(A46274). Accordingly, it is presently believed that PRO244 disclosed inthe present application is a newly identified member of the C-lectinsuperfamily and may play a role in immune function, apoptosis, or in thepathogenesis of atherosclerosis. In addition, PRO244 may be useful inidentifying tumor-associated epitopes.

B. PRO Polypeptide Variants

In addition to the full-length native sequence PRO polypeptidesdescribed herein, it is contemplated that PRO variants can be prepared.PRO variants can be prepared by introducing appropriate nucleotidechanges into the PRO DNA, and/or by synthesis of the desired PROpolypeptide. Those skilled in the art will appreciate that amino acidchanges may alter post-translational processes of the PRO, such aschanging the number or position of glycosylation sites or altering themembrane anchoring characteristics.

Variations in the native full-length sequence PRO or in various domainsof the PRO described herein, can be made, for example, using any of thetechniques and guidelines for conservative and non-conservativemutations set forth, for instance, in U.S. Pat. No. 5,364,934.Variations may be a substitution, deletion or insertion of one or morecodons encoding the PRO that results in a change in the amino acidsequence of the PRO as compared with the native sequence PRO. Optionallythe variation is by substitution of at least one amino acid with anyother amino acid in one or more of the domains of the PRO. Guidance indetermining which amino acid residue may be inserted, substituted ordeleted without adversely affecting the desired activity may be found bycomparing the sequence of the PRO with that of homologous known proteinmolecules and minimizing the number of amino acid sequence changes madein regions of high homology. Amino acid substitutions can be the resultof replacing one amino acid with another amino acid having similarstructural and/or chemical properties, such as the replacement of aleucine with a serine, i.e., conservative amino acid replacements.Insertions or deletions may optionally be in the range of about 1 to 5amino acids. The variation allowed may be determined by systematicallymaking insertions, deletions or substitutions of amino acids in thesequence and testing the resulting variants for activity exhibited bythe full-length or mature native sequence.

PRO polypeptide fragments are provided herein. Such fragments may betruncated at the N-terminus or C-terminus, or may lack internalresidues, for example, when compared with a full length native protein.Certain fragments lack amino acid residues that are not essential for adesired biological activity of the PRO polypeptide.

PRO fragments may be prepared by any of a number of conventionaltechniques. Desired peptide fragments may be chemically synthesized. Analternative approach involves generating PRO fragments by enzymaticdigestion, e.g., by treating the protein with an enzyme known to cleaveproteins at sites defined by particular amino acid residues, or bydigesting the DNA with suitable restriction enzymes and isolating thedesired fragment. Yet another suitable technique involves isolating andamplifying a DNA fragment encoding a desired polypeptide fragment, bypolymerase chain reaction (PCR). Oligonucleotides that define thedesired termini of the DNA fragment are employed at the 5′ and 3′primers in the PCR. Preferably, PRO polypeptide fragments share at leastone biological and/or immunological activity with the native PROpolypeptide disclosed herein.

In particular embodiments, conservative substitutions of interest areshown in Table 6 under the heading of preferred substitutions. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated exemplary substitutions in Table 6, oras further described below in reference to amino acid classes, areintroduced and the products screened.

TABLE 6 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his;lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) aspasp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu;val; met; ala; phe; leu norleucine Leu (L) norleucine; ile; val; ilemet; ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe(F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T)ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile;leu; met; phe; leu ala; norleucine

Substantial modifications in function or immunological identity of thePRO polypeptide are accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

-   (1) hydrophobic: norleucine, met, ala, val, leu, ile;-   (2) neutral hydrophilic: cys, ser, thr;-   (3) acidic: asp, glu;-   (4) basic: asn, gln, his, lys, arg;-   (5) residues that influence chain orientation: gly, pro; and-   (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the PRO variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant [Cunningham and Wells,Science, 244: 1081-1085 (1989)]. Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions [Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

C. Modifications of PRO

Covalent modifications of PRO are included within the scope of thisinvention. One type of covalent modification includes reacting targetedamino acid residues of a PRO polypeptide with an organic derivatizingagent that is capable of reacting with selected side chains or the N- orC-terminal residues of the PRO. Derivatization with bifunctional agentsis useful, for instance, for crosslinking PRO to a water-insolublesupport matrix or surface for use in the method for purifying anti-PROantibodies, and vice-versa. Commonly used crosslinking agents include,e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the PRO polypeptide includedwithin the scope of this invention comprises altering the nativeglycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence PRO (eitherby removing the underlying glycosylation site or by deleting theglycosylation by chemical and/or enzymatic means), and/or adding one ormore glycosylation sites that are not present in the native sequencePRO. In addition, the phrase includes qualitative changes in theglycosylation of the native proteins, involving a change in the natureand proportions of the various carbohydrate moieties present.

Addition of glycosylation sites to the PRO polypeptide may beaccomplished by altering the amino acid sequence. The alteration may bemade, for example, by the addition of, or substitution by, one or moreserine or threonine residues to the native sequence PRO (for 0-linkedglycosylation sites). The PRO amino acid sequence may optionally bealtered through changes at the DNA level, particularly by mutating theDNA encoding the PRO polypeptide at preselected bases such that codonsare generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on thePRO polypeptide is by chemical or enzymatic coupling of glycosides tothe polypeptide. Such methods are described in the art, e.g., in WO87/05330 published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit.Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the PRO polypeptide may beaccomplished chemically or enzymatically or by mutational substitutionof codons encoding for amino acid residues that serve as targets forglycosylation. Chemical deglycosylation techniques are known in the artand described, for instance, by Hakimuddin, et al., Arch. Biochem.Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptides canbe achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., Meth. Enzymol., 138:350 (1987).

Another type of covalent modification of PRO comprises linking the PROpolypeptide to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, inthe manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

The PRO of the present invention may also be modified in a way to form achimeric molecule comprising PRO fused to another, heterologouspolypeptide or amino acid sequence.

In one embodiment, such a chimeric molecule comprises a fusion of thePRO with a tag polypeptide which provides an epitope to which ananti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the PRO. The presence ofsuch epitope-tagged forms of the PRO can be detected using an antibodyagainst the tag polypeptide. Also, provision of the epitope tag enablesthe PRO to be readily purified by affinity purification using ananti-tag antibody or another type of affinity matrix that binds to theepitope tag. Various tag polypeptides and their respective antibodiesare well known in the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science 255:192-194 (1992)]; an α-tubulin epitope peptide [Skinneret al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10protein peptide tag [Lutz-Freyemuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)].

In an alternative embodiment, the chimeric molecule may comprise afusion of the PRO with an immunoglobulin or a particular region of animmunoglobulin. For a bivalent form of the chimeric molecule (alsoreferred to as an “immunoadhesin”), such a fusion could be to the Fcregion of an IgG molecule. The Ig fusions preferably include thesubstitution of a soluble (transmembrane domain deleted or inactivated)form of a PRO polypeptide in place of at least one variable regionwithin an Ig molecule. In a particularly preferred embodiment, theimmunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge,CH1, CH2 and CH3 regions of an IgG1 molecule. For the production ofimmunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27,1995.

D. Preparation of PRO

The description below relates primarily to production of PRO byculturing cells transformed or transfected with a vector containing PROnucleic acid. It is, of course, contemplated that alternative methods,which are well known in the art, may be employed to prepare PRO. Forinstance, the PRO sequence, or portions thereof, may be produced bydirect peptide synthesis using solid-phase techniques [see, e.g.,Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., SanFrancisco, Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154(1963)]. In vitro protein synthesis may be performed using manualtechniques or by automation. Automated synthesis may be accomplished,for instance, using an Applied Biosystems Peptide Synthesizer (FosterCity, Calif.) using manufacturer's instructions. Various portions of thePRO may be chemically synthesized separately and combined using chemicalor enzymatic methods to produce the full-length PRO.

-   -   1. Isolation of DNA Encoding PRO

DNA encoding PRO may be obtained from a cDNA library prepared fromtissue believed to possess the PRO mRNA and to express it at adetectable level. Accordingly, human PRO DNA can be convenientlyobtained from a cDNA library prepared from human tissue, such asdescribed in the Examples. The PRO-encoding gene may also be obtainedfrom a genomic library or by known synthetic procedures (e.g., automatednucleic acid synthesis).

Libraries can be screened with probes (such as antibodies to the PRO oroligonucleotides of at least about 20-80 bases) designed to identify thegene of interest or the protein encoded by it. Screening the cDNA orgenomic library with the selected probe may be conducted using standardprocedures, such as described in Sambrook et al., Molecular Cloning: ALaboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).An alternative means to isolate the gene encoding PRO is to use PCRmethodology [Sambrook et al., supra; Dieffenbach et al., PCR Primer: ALaboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].

The Examples below describe techniques for screening a cDNA library. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra.

Sequences identified in such library screening methods can be comparedand aligned to other known sequences deposited and available in publicdatabases such as GenBank or other private sequence databases. Sequenceidentity (at either the amino acid or nucleotide level) within definedregions of the molecule or across the full-length sequence can bedetermined using methods known in the art and as described herein.

Nucleic acid having protein coding sequence may be obtained by screeningselected cDNA or genomic libraries using the deduced amino acid sequencedisclosed herein for the first time, and, if necessary, usingconventional primer extension procedures as described in Sambrook etal., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

-   -   2. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for PRO production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.The culture conditions, such as media, temperature, pH and the like, canbe selected by the skilled artisan without undue experimentation. Ingeneral, principles, protocols, and practical techniques for maximizingthe productivity of cell cultures can be found in Mammalian CellBiotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991)and Sambrook et al., supra.

Methods of eukaryotic cell transfection and prokaryotic celltransformation are known to the ordinarily skilled artisan, for example,CaCl₂, CaPO₄, liposome-mediated and electroporation. Depending on thehost cell used, transformation is performed using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in Sambrook et al., supra, or electroporation isgenerally used for prokaryotes. Infection with Agrobacterium tumefaciensis used for transformation of certain plant cells, as described by Shawet al., Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989.Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransfections have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E.coli strain W3110 (ATCC 27,325) and K5772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting. Strain W3110 is one particularly preferred host or parenthost because it is a common host strain for recombinant DNA productfermentations. Preferably, the host cell secretes minimal amounts ofproteolytic enzymes. For example, strain W3110 may be modified to effecta genetic mutation in the genes encoding proteins endogenous to thehost, with examples of such hosts including E. coli W3110 strain 1A2,which has the complete genotype tonA; E. coli W3110 strain 9E4, whichhas the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC55,244), which has the complete genotype tonA ptr3phoA E15 (argF-lac)169degP ompTkan^(r) ; E. coli W3110 strain 37D6, which has the completegenotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7 ilvG kan^(r) ;E. coli W3110 strain 40B4, which is strain 37D6 with a non-kanamycinresistant degP deletion mutation; and an E. coli strain having mutantperiplasmic protease disclosedin U.S. Pat. No. 4,946,783 issued 7 Aug.1990. Alternatively, in vitro methods of cloning, e.g., PCR or othernucleic acid polymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for PRO-encodingvectors. Saccharomyces cerevisiae is a commonly used lower eukaryotichost microorganism. Others include Schizosaccharomyces pombe (Beach andNurse, Nature, 290: 140 [1981]; EP 139,383 published May 2, 1985);Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al.,Bio/Technology, 9:968-975 (1991)) such as, e.g., K. lactis (MW98-8C,CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 737 [1983]), K.fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van denBerg et al., Bio/Technology, 8:135 (1990)), K. thermotolerans, and K.marxianus; yarrowia EP 402,226); Pichia pastoris (EP 183,070;Sreekrishna et al., J. Basic Microbiol., 28:265-278 [1988]); Candida;Trichoderna reesia (EP 244,234); Neurospora crassa (Case et al., Proc.Natl. Acad. Sci. USA, 76:5259-5263 [1979]); Schwanniomyces such asSchwannionyces occidentalis (EP 394,538 published Oct. 31, 1990); andfilamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium(WO 91/00357 published Jan. 10, 1991), and Aspergillus hosts such as A.nidulans (3allance et al., Biochem. Biophys. Res. Commun., 112:284-289[1983]; Tilburn et al., Gene, 26:205-221 [1983]; Yelton et al., Proc.Natl. Acad. Sci. USA, 81: 1470-1474 [1984]) and A. niger (Kelly andHynes, EMBO J., 4:475-479 [1985]). Methylotropic yeasts are suitableherein and include, but are not limited to, yeast capable of growth onmethanol selected from the genera consisting of Hansenula, Candida,Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list ofspecific species that are exemplary of this class of yeasts may be foundin C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).

Suitable host cells for the expression of glycosylated PRO are derivedfrom multicellular organisms. Examples of invertebrate cells includeinsect cells such as Drosophila S2 and Spodoptera Sf9, as well as plantcells. Examples of useful mammalian host cell lines include Chinesehamster ovary (CHO) and COS cells. More specific examples include monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); Chinesehamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad.Sci. USA, 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol.Reprod., 23:243-251(1980)); human lung cells (W138, ATCC CCL 75); humanliver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562, ATCCCCL51). The selection of the appropriate host cell is deemed to bewithin the skill in the art.

-   -   3. Selection and Use of a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO may beinserted into a replicable vector for cloning (amplification of the DNA)or for expression. Various vectors are publicly available. The vectormay, for example, be in the form of a plasmid, cosmid, viral particle,or phage. The appropriate nucleic acid sequence may be inserted into thevector by a variety of procedures. In general, DNA is inserted into anappropriate restriction endonuclease site(s) using techniques known inthe art. Vector components generally include, but are not limited to,one or more of a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence. Construction of suitable vectors containing one ormore of these components employs standard ligation techniques which areknown to the skilled artisan.

The PRO may be produced recombinantly not only directly, but also as afusion polypeptide with a heterologous polypeptide, which may be asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. In general, thesignal sequence may be a component of the vector, or it may be a part ofthe PRO-encoding DNA that is inserted into the vector. The signalsequence may be a prokaryotic signal sequence selected, for example,from the group of the alkaline phosphatase, penicillinase, lpp, orheat-stable enterotoxin II leaders. For yeast secretion the signalsequence may be, e.g., the yeast invertase leader, alpha factor leader(including Saccharomyces and Kluyveromyces α-factor leaders, the latterdescribed in U.S. Pat. No. 5,010,182), or acid phosphatase leader, theC. albicans glucoamylase leader (EP 362,179 published 4 Apr 1990), orthe signal described in WO 90/13646 published 15 Nov. 1990. In mammaliancell expression, mammalian signal sequences may be used to directsecretion of the protein, such as signal sequences from secretedpolypeptides of the same or related species, as well as viral secretoryleaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGrain-negative bacteria, the 2 μ plasmid origin is suitable for yeast,and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) areuseful for cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up thePRO-encoding nucleic acid, such as DHFR or thymidine kinase. Anappropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitableselection gene for use in yeast is the trp1 gene present in the yeastplasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1[Jones, Genetics, , 85:12 (1977)].

Expression and cloning vectors usually contain a promoter operablylinked to the PRO-encoding nucleic acid sequence to direct mRNAsynthesis. Promoters recognized by a variety of potential host cells arewell known. Promoters suitable for use with prokaryotic hosts includethe β-lactamase and lactose promoter systems [Chang et al., Nature,275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkalinephosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic AcidsRes., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tacpromoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)].Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding PRO.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexolinase, pyruvate decarboxylase, phosphofructokinase,glucose-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase,triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

PRO transcription from vectors in mammalian host cells is controlled,for example, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989),adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus40 (SV40), from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, and from heat-shock promoters,provided such promoters are compatible with the host cell systems.

Transcription of a DNA encoding the PRO by higher eukaryotes may beincreased by inserting an enhancer sequence into the vector. Enhancersare cis-acting elements of DNA, usually about from 10 to 300 bp, thatact on a promoter to increase its transcription. Many enhancer sequencesare now known from mammalian genes (globin, elastase, albumin,α-fetoprotein, and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to thePRO coding sequence, but is preferably located at a site 5′ from thepromoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding PRO.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of PRO in recombinant vertebrate cell culture aredescribed in Gething et al., Nature, 293:620-625 (1981); Mantei et al.,Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.

-   -   4. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by iununologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequencePRO polypeptide or against a synthetic peptide based on the DNAsequences provided herein or against exogenous sequence fused to PRO DNAand encoding a specific antibody epitope.

-   -   5. Purification of Polypeptide

Forms of PRO may be recovered from culture medium or from host celllysates. If membrane-bound, it can be released from the membrane using asuitable detergent solution (e.g. Triton-X 100) or by enzymaticcleavage. Cells employed in expression of PRO can be disrupted byvarious physical or chemical means, such as freeze-thaw cycling,sonication, mechanical disruption, or cell lysing agents.

It may be desired to purify PRO from recombinant cell proteins orpolypeptides. The following procedures are exemplary of suitablepurification procedures: by fractionation on an ion-exchange column;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; gel filtration using, for example,Sephadex G-75; protein A Sepharose columns to remove contaminants suchas IgG; and metal chelating columns to bind epitope-tagged forms of thePRO. Various methods of protein purification may be employed and suchmethods are known in the art and described for example in Deutscher,Methods in Enzymology, 182 (1990); Scopes, Protein Purification:Principles and Practice, Springer-Verlag, New York (1982). Thepurification step(s) selected will depend, for example, on the nature ofthe production process used and the particular PRO produced.

E. Uses for PRO

Nucleotide sequences (or their complement) encoding PRO have variousapplications in the art of molecular biology, including uses ashybridization probes, in chromosome and gene mapping and in thegeneration of anti-sense RNA and DNA. PRO nucleic acid will also beuseful for the preparation of PRO polypeptides by the recombinanttechniques described herein.

The full-length native sequence PRO gene, or portions thereof, may beused as hybridization probes for a cDNA library to isolate thefull-length PRO cDNA or to isolate still other cDNAs (for instance,those encoding naturally-occurring variants of PRO or PRO from otherspecies) which have a desired sequence identity to the native PROsequence disclosed herein. Optionally, the length of the probes will beabout 20 to about 50 bases. The hybridization probes may be derived fromat least partially novel regions of the full length native nucleotidesequence wherein those regions may be determined without undueexperimentation or from genomic sequences including promoters, enhancerelements and introns of native sequence PRO. By way of example, ascreening method will comprise isolating the coding region of the PROgene using the known DNA sequence to synthesize a selected probe ofabout 40 bases. Hybridization probes may be labeled by a variety oflabels, including radionucleotides such as ³²P or 35S, or enzymaticlabels such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems. Labeled probes having a sequencecomplementary to that of the PRO gene of the present invention can beused to screen libraries of human cDNA, genomic DNA or mRNA to determinewhich members of such libraries the probe hybridizes to. Hybridizationtechniques are described in further detail in the Examples below.

Any EST sequences disclosed in the present application may similarly beemployed as probes, using the methods disclosed herein.

Other useful fragments of the PRO nucleic acids include antisense orsense oligonucleotides comprising a singe-stranded nucleic acid sequence(either RNA or DNA) capable of binding to target PRO mRNA (sense) or PRODNA (antisense) sequences. Antisense or sense oligonucleotides,according to the present invention, comprise a fragment of the codingregion of PRO DNA. Such a fragment generally comprises at least about 14nucleotides, preferably from about 14 to 30 nucleotides. The ability toderive an antisense or a sense oligonucleotide, based upon a cDNAsequence encoding a given protein is described in, for example, Steinand Cohen (Cancer Res. . 48:2659, 1988) and van der Krol et al.(BioTechniques 6:958, 1988).

Binding of antisense or sense oligonucleotides to target nucleic acidsequences results in the formation of duplexes that block transcriptionor translation of the target sequence by one of several means, includingenhanced degradation of the duplexes, premature termination oftranscription or translation, or by other means. The antisenseoligonucleotides thus may be used to block expression of PRO proteins.Antisense or sense oligonucleotides further comprise oligonucleotideshaving modified sugar-phosphodiester backbones (or other sugar linkages,such as those described in WO 91/06629) and wherein such sugar linkagesare resistant to endogenous nucleases. Such oligonucleotides withresistant sugar linkages are stable in vivo (i.e., capable of resistingenzymatic degradation) but retain sequence specificity to be able tobind to target nucleotide sequences.

Other examples of sense or antisense oligonucleotides include thoseoligonucleotides which are covalently linked to organic moieties, suchas those described in WO 90/10048, and other moieties that increasesaffinity of the oligonucleotide for a target nucleic acid sequence, suchas poly-(L-lysine). Further still, intercalating agents, such asellipticine, and alkylating agents or metal complexes may be attached tosense or antisense oligonucleotides to modify binding specificities ofthe antisense or sense oligonucleotide for the target nucleotidesequence.

Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. In a preferred procedure, an antisense or sense oligonucleotideis inserted into a suitable retroviral vector. A cell containing thetarget nucleic acid sequence is contacted with the recombinantretroviral vector, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, those derived from the murineretrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the doublecopy vectors designated DCT5A, DCT5B and DCT5C (see WO 90/13641).

Sense or antisense oligonucleotides also may be introduced into a cellcontaining the target nucleotide sequence by formation of a conjugatewith a ligand binding molecule, as described in WO 91/04753. Suitableligand binding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense oligonucleotide or its conjugatedversion into the cell.

Alternatively, a sense or an antisense oligonucleotide may be introducedinto a cell containing the target nucleic acid sequence by formation ofan oligonucleotide-lipid complex, as described in WO 90/10448. The senseor antisense oligonucleotide-lipid complex is preferably dissociatedwithin the cell by an endogenous lipase.

Antisense RNA or DNA molecules are generally at least about 5 bases inlength, about 10 bases in length, about 15 bases in length, about 20bases in length, about 25 bases in length, about 30 bases in length,about 35 bases in length, about 40 bases in length, about 45 bases inlength, about 50 bases in length, about 55 bases in length, about 60bases in length, about 65 bases in length, about 70 bases in length,about 75 bases in length, about 80 bases in length, about 85 bases inlength, about 90 bases in length, about 95 bases in length, about 100bases in length, or more.

The probes may also be employed in PCR techniques to generate a pool ofsequences for identification of closely related PRO coding sequences.

Nucleotide sequences encoding a PRO can also be used to constructhybridization probes for mapping the gene which encodes that PRO and forthe genetic analysis of individuals with genetic disorders. Thenucleotide sequences provided herein may be mapped to a chromosome andspecific regions of a chromosome using known techniques, such as in situhybridization, linkage analysis against known chromosomal markers, andhybridization screening with libraries.

When the coding sequences for PRO encode a protein which binds toanother protein (example, where the PRO is a receptor), the PRO can beused in assays to identify the other proteins or molecules involved inthe binding interaction. By such methods, inhibitors of thereceptor/ligand binding interaction can be identified. Proteins involvedin such binding interactions can also be used to screen for peptide orsmall molecule inhibitors or agonists of the binding interaction. Also,the receptor PRO can be used to isolate correlative ligand(s). Screeningassays can be designed to find lead compounds that mimic the biologicalactivity of a native PRO or a receptor for PRO. Such screening assayswill include assays amenable to high-throughput screening of chemicallibraries, making them particularly suitable for identifying smallmolecule drug candidates. Small molecules contemplated include syntheticorganic or inorganic compounds. The assays can be performed in a varietyof formats, including protein-protein binding assays, biochemicalscreening assays, immunoassays and cell based assays, which are wellcharacterized in the art.

Nucleic acids which encode PRO or its modified forms can also be used togenerate either transgenic animals or “knock outs animals which, inturn, are useful in the development and screening of therapeuticallyuseful reagents. A transgenic animal (e.g., a mouse or rat) is an animalhaving cells that contain a transgene, which transgene was introducedinto the animal or an ancestor of the animal at a prenatal, e.g., anembryonic stage. A transgene is a DNA which is integrated into thegenome of a cell from which a transgenic animal develops. In oneembodiment, cDNA encoding PRO can be used to clone genomic DNA encodingPRO in accordance with established techniques and the genomic sequencesused to generate transgenic animals that contain cells which express DNAencoding PRO. Methods for generating transgenic animals, particularlyanimals such as mice or rats, have become conventional in the art andare described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009.Typically, particular cells would be targeted for PRO transgeneincorporation with tissue-specific enhancers. Transgenic animals thatinclude a copy of a transgene encoding PRO introduced into the germ lineof the animal at an embryonic stage can be used to examine the effect ofincreased expression of DNA encoding PRO. Such animals can be used astester animals for reagents thought to confer protection from, forexample, pathological conditions associated with its overexpression. Inaccordance with this facet of the invention, an animal is treated withthe reagent and a reduced incidence of the pathological condition,compared to untreated animals bearing the transgene, would indicate apotential therapeutic intervention for the pathological condition.

Alternatively, non-human homologues of PRO can be used to construct aPRO “knock out” animal which has a defective or altered gene encodingPRO as a result of homologous recombination between the endogenous geneencoding PRO and altered genomic DNA encoding PRO introduced into anembryonic stem cell of the animal. For example, cDNA encoding PRO can beused to clone genomic DNA encoding PRO in accordance with establishedtechniques. A portion of the genomic DNA encoding PRO can be deleted orreplaced with another gene, such as a gene encoding a selectable markerwhich can be used to monitor integration. Typically, several kilobasesof unaltered flanking DNA (both at the 5′ and 3′ ends) are included inthe vector [see e.g., Thomas and Capecchi, Cell, 51:503 (1987) for adescription of homologous recombination vectors]. The vector isintroduced into an embryonic stem cell line (e.g., by electroporation)and cells in which the introduced DNA has homologously recombined withthe endogenous DNA are selected [see e.g., Li et al., Cell, 69:915(1992)]. The selected cells are then injected into a blastocyst of ananimal (e.g., a mouse or rat) to form aggregation chimeras [see e.g.,Bradley, in Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. Achimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term to create a “knockout” animal. Progeny harboring the homologously recombined DNA in theirgerm cells can be identified by standard techniques and used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA. Knockout animals can be characterized for instance, fortheir ability to defend against certain pathological conditions and fortheir development of pathological conditions due to absence of the PROpolypeptide.

Nucleic acid encoding the PRO polypeptides may also be used in genetherapy. In gene therapy applications, genes are introduced into cellsin order to achieve in vivo synthesis of a therapeutically effectivegenetic product, for example for replacement of a defective gene. “Genetherapy” includes both conventional gene therapy where a lasting effectis achieved by a single treatment, and the administration of genetherapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA. AntisenseRNAs and DNAs can be used as therapeutic agents for blocking theexpression of certain genes in vivo. It has already been shown thatshort antisense oligonucleotides can be imported into cells where theyact as inhibitors, despite their low intracellular concentrations causedby their restricted uptake by the cell membrane. (Zamecnik et al., Proc.Natl. Acad. Sci. USA 83:4143-4146 [1986]). The oligonucleotides can bemodified to enhance their uptake, e.g. by substituting their negativelycharged phosphodiester groups by uncharged groups.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. The currently preferred in vivogene transfer techniques include transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection (Dzau et al., Trends in Biotechnology 11, 205-210 [1993]).In some situations it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA 87, 3410-3414 (1990). For review of gene marking and genetherapy protocols see Anderson et al., Science 256, 808-813 (1992).

The PRO polypeptides described herein may also be employed as molecularweight markers for protein electrophoresis purposes and the isolatednucleic acid sequences may be used for recombinantly expressing thosemarkers

The nucleic acid molecules encoding the PRO polypeptides or fragmentsthereof described herein are useful for chromosome identification. Inthis regard, there exists an ongoing need to identify new chromosomemarkers, since relatively few chromosome marking reagents, based uponactual sequence data are presently available. Each PRO nucleic acidmolecule of the present invention can be used as a chromosome marker.

The PRO polypeptides and nucleic acid molecules of the present inventionmay also be used for tissue typing, wherein the PRO polypeptides of thepresent invention may be differentially expressed in one tissue ascompared to another. PRO nucleic acid molecules will find use forgenerating probes for PCR, Northern analysis, Southern analysis andWestern analysis.

The PRO polypeptides described herein may also be employed astherapeutic agents. The PRO polypeptides of the present invention can beformulated according to known methods to prepare pharmaceutically usefulcompositions, whereby the PRO product hereof is combined in admixturewith a pharmaceutically acceptable carrier vehicle. Therapeuticformulations are prepared for storage by mixing the active ingredienthaving the desired degree of purity with optional physiologicallyacceptable carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Acceptable carriers,excipients or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrateand other organic acids; antioxidants including ascorbic acid; lowmolecular weight (less than about 10 residues) polypeptides; proteins,such as serum albumin, gelatin or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidone, amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides and othercarbohydrates including glucose, mannose, or dextrins; chelating agentssuch as EDTA; sugar alcohols such as mannitol or sorbitol; salt-formingcounterions such as sodium; and/or nonionic surfactants such as TWEEN™,PLURONICS™ or PEG.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to or following lyophilization and reconstitution.

Therapeutic compositions herein generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is in accord with known methods, e.g.injection or infusion by intravenous, intraperitoneal, intracerebral,intramuscular, intraocular, intraarterial or intralesional routes,topical administration, or by sustained release systems.

Dosages and desired drug concentrations of pharmaceutical compositionsof the present invention may vary depending on the particular useenvisioned. The determination of the appropriate dosage or route ofadministration is well within the skill of an ordinary physician. Animalexperiments provide reliable guidance for the determination of effectivedoses for human therapy. Interspecies scaling of effective doses can beperformed following the principles laid down by Mordenti, J. andChappell, W. “The use of interspecies scaling in toxicokinetics” InToxicokinetics and New Drug Development, Yacobi et al., Eds., PergamonPress, New York 1989, pp. 42-96.

When in vivo administration of a PRO polypeptide or agonist orantagonist thereof is employed, normal dosage amounts may vary fromabout 10 ng/kg to up to 100 mg/kg of mammal body weight or more per day,preferably about 1 μg/kg/day to 10 mg/kg/day, depending upon the routeof administration. Guidance as to particular dosages and methods ofdelivery is provided in the literature; see, for example, U.S. Pat. Nos.4,657,760; 5,206,344; or 5,225,212. It is anticipated that differentformulations will be effective for different treatment compounds anddifferent disorders, that administration targeting one organ or tissue,for example, may necessitate delivery in a manner different from that toanother organ or tissue.

Where sustained-release administration of a PRO polypeptide is desiredin a formulation with release characteristics suitable for the treatmentof any disease or disorder requiring administration of the PROpolypeptide, microencapsulation of the PRO polypeptide is contemplated.Microencapsulation of recombinant proteins for sustained release hasbeen successfully performed with human growth hormone (rhGH),interferon-(rhIFN-), interleukin-2, and MN rgp120. Johnson et al., Nat.Med., 2:795-799 (1996); Yasuda, Biomed. Ther., 27:1221-1223 (1993); Horaet al., Bio/Technologs, 8:755-758 (1990); Cleland, “Design and Produc ofSingle Immunization Vaccines Using Polylactide Polyglycolide MicrosphereSystems,” in Vaccine Design: The Subunit and Adjuvant Approach, Powelland Newman, eds, (Plenum Press: New York, 1995), pp. 439-462; WO97/03692, WO 96/40072, WO 96/07399; and U.S. Pat. No. 5,654,010.

The sustained-release formulations of these proteins were developedusing poly-lactic-coglycolic acid (PLGA) polymer due to itsbiocompatibility and wide range of biodegradable properties. Thedegradation products of PLGA, lactic and glycolic acids, can be clearedquickly within the human body. Moreover, the degradability of thispolymer can be adjusted from months to years depending on its molecularweight and composition. Lewis, “Controlled release of bioactive agentsfrom lactide/glycolide polymer,” in: M. Chasin and R. Langer (Eds.),Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: NewYork, 1990), pp. 1-41.

This invention encompasses methods of screening compounds to identifythose that mimic the PRO polypeptide (agonists) or prevent the effect ofthe PRO polypeptide (antagonists). Screening assays for antagonist drugcandidates are designed to identify compounds that bind or complex withthe PRO polypeptides encoded by the genes identified herein, orotherwise interfere with the interaction of the encoded polypeptideswith other cellular proteins. Such screening assays will include assaysamenable to high-throughput screening of chemical libraries, making themparticularly suitable for identifying small molecule drug candidates.

The assays can be performed in a variety of formats, includingprotein-protein binding assays, biochemical screening assays,immunoassays, and cell-based assays, which are well characterized in theart.

All assays for antagonists are common in that they call for contactingthe drug candidate with a PRO polypeptide encoded by a nucleic acididentified herein under conditions and for a time sufficient to allowthese two components to interact.

In binding assays, the interaction is binding and the complex formed canbe isolated or detected in the reaction mixture. In a particularembodiment, the PRO polypeptide encoded by the gene identified herein orthe drug candidate is immobilized on a solid phase, e.g., on amicrotiter plate, by covalent or non-covalent attachments. Non-covalentattachment generally is accomplished by coating the solid surface with asolution of the PRO polypeptide and drying. Alternatively, animmobilized antibody, e.g., a monoclonal antibody, specific for the PROpolypeptide to be immobilized can be used to anchor it to a solidsurface. The assay is performed by adding the non-immobilized component,which may be labeled by a detectable label, to the immobilizedcomponent, e.g., the coated surface containing the anchored component.When the reaction is complete, the non-reacted components are removed,e.g., by washing, and complexes anchored on the solid surface aredetected. When the originally non-immobilized component carries adetectable label, the detection of label immobilized on the surfaceindicates that complexing occurred. Where the originally non-immobilizedcomponent does not carry a label, complexing can be detected, forexample, by using a labeled antibody specifically binding theimmobilized complex.

If the candidate compound interacts with but does not bind to aparticular PRO polypeptide encoded by a gene identified herein, itsinteraction with that polypeptide can be assayed by methods well knownfor detecting protein-protein interactions. Such assays includetraditional approaches, such as, e.g., cross-linking,co-immunoprecipitation, and co-purification through gradients orchromatographic columns. In addition, protein-protein interactions canbe monitored by using a yeast-based genetic system described by Fieldsand co-workers (Fields and Song, Nature (London), 340:245-246 (1989);Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578-9582 (1991)) asdisclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89:5789-5793 (1991). Many transcriptional activators, such as yeast GAL4,consist of two physically discrete modular domains, one acting as theDNA-binding domain, the other one functioning as thetranscription-activation domain. The yeast expression system describedin the foregoing publications (generally referred to as the “two-hybridsystem”) takes advantage of this property, and employs two hybridproteins, one in which the target protein is fused to the DNA-bindingdomain of GAL4, and another, in which candidate activating proteins arefused to the activation domain. The expression of a GAL1-lacZ reportergene under control of a GAL4-activated promoter depends onreconstitution of GAL4 activity via protein-protein interaction.Colonies containing interacting polypeptides are detected with achromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™)for identifying protein-protein interactions between two specificproteins using the two-hybrid technique is commercially available fromClontech. This system can also be extended to map protein domainsinvolved in specific protein interactions as well as to pinpoint aminoacid residues that are crucial for these interactions.

Compounds that interfere with the interaction of a gene encoding a PROpolypeptide identified herein and other intra- or extracellularcomponents can be tested as follows: usually a reaction mixture isprepared containing the product of the gene and the intra- orextracellular component under conditions and for a time allowing for theinteraction and binding of the two products. To test the ability of acandidate compound to inhibit binding, the reaction is run in theabsence and in the presence of the test compound. In addition, a placebomay be added to a third reaction mixture, to serve as positive control.The binding (complex formation) between the test compound and the intra-or extracellular component present in the mixture is monitored asdescribed hereinabove. The formation of a complex in the controlreaction(s) but not in the reaction mixture containing the test compoundindicates that the test compound interferes with the interaction of thetest compound and its reaction partner.

To assay for antagonists, the PRO polypeptide may be added to a cellalong with the compound to be screened for a particular activity and theability of the compound to inhibit the activity of interest in thepresence of the PRO polypeptide indicates that the compound is anantagonist to the PRO polypeptide. Alternatively, antagonists may bedetected by combining the PRO polypeptide and a potential antagonistwith membrane-bound PRO polypeptide receptors or recombinant receptorsunder appropriate conditions for a competitive inhibition assay. The PROpolypeptide can be labeled, such as by radioactivity, such that thenumber of PRO polypeptide molecules bound to the receptor can be used todetermine the effectiveness of the potential antagonist. The geneencoding the receptor can be identified by numerous methods known tothose of skill in the art, for example, ligand panning and FACS sorting.Coligan et al., Current Protocols in Immun., 1(2): Chapter 5 (1991).Preferably, expression cloning is employed wherein polyadenylated RNA isprepared from a cell responsive to the PRO polypeptide and a cDNAlibrary created from this RNA is divided into pools and used totransfect COS cells or other cells that are not responsive to the PROpolypeptide. Transfected cells that are grown on glass slides areexposed to labeled PRO polypeptide. The PRO polypeptide can be labeledby a variety of means including iodination or inclusion of a recognitionsite for a site-specific protein kinase. Following fixation andincubation, the slides are subjected to autoradiographic analysis.Positive pools are identified and sub-pools are prepared andre-transfected using an interactive sub-pooling and re-screeningprocess, eventually yielding a single clone that encodes the putativereceptor.

As an alternative approach for receptor identification, labeled PROpolypeptide can be photoaffinity-linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE and exposed to X-ray film. The labeled complexcontaining the receptor can be excised, resolved into peptide fragments,and subjected to protein micro-sequencing. The amino acid sequenceobtained from micro-sequencing would be used to design a set ofdegenerate oligonucleotide probes to screen a cDNA library to identifythe gene encoding the putative receptor.

In another assay for antagonists, mammalian cells or a membranepreparation expressing the receptor would be incubated with labeled PROpolypeptide in the presence of the candidate compound. The ability ofthe compound to enhance or block this interaction could then bemeasured.

More specific examples of potential antagonists include anoligonucleotide that binds to the fusions of immunoglobulin with PROpolypeptide, and, in particular, antibodies including, withoutlimitation, poly- and monoclonal antibodies and antibody fragments,single-chain antibodies, anti-idiotypic antibodies, and chimeric orhumanized versions of such antibodies or fragments, as well as humanantibodies and antibody fragments. Alternatively, a potential antagonistmay be a closely related protein, for example, a mutated form of the PROpolypeptide that recognizes the receptor but imparts no effect, therebycompetitively inhibiting the action of the PRO polypeptide.

Another potential PRO polypeptide antagonist is an antisense RNA or DNAconstruct prepared using antisense technology, where, e.g., an antisenseRNA or DNA molecule acts to block directly the translation of mRNA byhybridizing to targeted mRNA and preventing protein translation.Antisense technology can be used to control gene expression throughtriple-helix formation or antisense DNA or RNA, both of which methodsare based on binding of a polynucleotide to DNA or RNA. For example, the5′ coding portion of the polynucleotide sequence, which encodes themature PRO polypeptides herein, is used to design an antisense RNAoligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix—see Lee et al., Nucl. AcidsRes., 6:3073 (1979); Cooney et al., Science, 241: 456 (1988); Dervan etal., Science, 251:1360 (1991)), thereby preventing transcription and theproduction of the PRO polypeptide. The antisense RNA oligonucleotidehybridizes to the mRNA in vivo and blocks translation of the mRNAmolecule into the PRO polypeptide (antisense—Okano, Neurochem., 56:560(1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression(CRC Press: Boca Raton, Fla., 1988). The oligonucleotides describedabove can also be delivered to cells such that the antisense RNA or DNAmay be expressed in vivo to inhibit production of the PRO polypeptide.When antisense DNA is used, oligodeoxyribonucleotides derived from thetranslation-initiation site, e.g., between about −10 and +10 positionsof the target gene nucleotide sequence, are preferred.

Potential antagonists include small molecules that bind to the activesite, the receptor binding site, or growth factor or other relevantbinding site of the PRO polypeptide, thereby blocking the normalbiological activity of the PRO polypeptide. Examples of small moleculesinclude, but are not limited to, small peptides or peptide-likemolecules, preferably soluble peptides, and synthetic non-peptidylorganic or inorganic compounds.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. Ribozymes act by sequence-specific hybridization to thecomplementary target RNA, followed by endonucleolytic cleavage. Specificribozyme cleavage sites within a potential RNA target can be identifiedby known techniques. For further details see, e.g., Rossi, CurrentBiology, 4:469-471 (1994), and PCT publication No. WO 97/33551(published Sep. 18, 1997).

Nucleic acid molecules in triple-helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple-helix formation via Hoogsteenbase-pairing rules, which generally require sizeable stretches ofpurines or pyrimidines on one strand of a duplex. For further detailssee, e.g., PCT publication No. WO 97/33551, supra.

These small molecules can be identified by any one or more of thescreening assays discussed hereinabove and/or by any other screeningtechniques well known for those skilled in the art.

With regard to the PRO211 and PRO217 polypeptide, therapeuticindications include disorders associated with the preservation andmaintenance of gastrointestinal mucosa and the repair of acute andchronic mucosal lesions (e.g., enterocolitis, Zollinger-Ellisonsyndrome, gastrointestinal ulceration and congenital microvillusatrophy), skin diseases associated with abnormal keratinocytedifferentiation (e.g., psoriasis, epithelial cancers such as lungsquamous cell carcinoma, epidermoid carcinoma of the vulva and gliomas.

Since the PRO232 polypeptide and nucleic acid encoding it possesssequence homology to a cell surface stem cell antigen and its encodingnucleic acid, probes based upon the PRO232 nucleotide sequence may beemployed to identify other novel stem cell surface antigen proteins.Soluble forms of the PRO232 polypeptide may be employed as antagonistsof membrane bound PRO232 activity both in vitro and in vivo. PRO232polypeptides may be employed in screening assays designed to identifyagonists or antagonists of the native PRO232 polypeptide, wherein suchassays may take the form of any conventional cell-type or biochemicalbinding assay. Moreover, the PRO232 polypeptide may serve as a molecularmarker for the tissues in which the polypeptide is specificallyexpressed.

With regard to the PRO187 polypeptides disclosed herein, FGF-8 has beenimplicated in cellular differentiation and embryogenesis, including thepatterning which appears during limb formation. FGF-8 and the PRO187molecules of the invention therefore are likely to have potent effectson cell growth and development. Diseases which relate to cellular growthand differentiation are therefore suitable targets for therapeuticsbased on functionality similar to FGF-8. For example, diseases relatedto growth or survival of nerve cells including Parkinson's disease,Alzheimer's disease, ALS, neuropathies. Additionally, disease related touncontrolled cell growth, e.g., cancer, would also be expectedtherapeutic targets.

With regard to the PRO265 polypeptides disclosed herein, other methodsfor use with PRO265 are described in U.S. Pat. No. 5,654,270 toRuoslahti et al. In particular, PRO265 can be used in comparison withthe fibromodulin disclosed therein to compare its effects on reducingdermal scarring and other properties of the fibromodulin describedtherein including where it is located and with what it binds and doesnot.

The PRO219 polypeptides of the present invention which play a regulatoryrole in the blood coagulation cascade may be employed in vivo fortherapeutic purposes as well as for in vitro purposes. Those of ordinaryskill in the art will well know how to employ PRO219 polypeptides forsuch uses.

The PRO246 polypeptides of the present invention which serve as cellsurface receptors for one or more viruses will find other uses. Forexample, extracellular domains derived from these PRO246 polypeptidesmay be employed therapeutically in vivo for lessening the effects ofviral infection. Those PRO246 polypeptides which serves as tumorspecific antigens may be exploited as therapeutic targets for anti-tumordrugs, and the like. Those of ordinary skill in the art will well knowhow to employ PRO246 polypeptides for such uses.

Assays in which connective growth factor and other growth factors areusually used should be performed with PRO261. An assay to determinewhether TGF beta induces PRO261, indicating a role in cancer isperformed as known in the art. Wound repair and tissue growth assays arealso performed with PRO261. The results are applied accordingly.

PRO228 polypeptides should be used in assays in which EMR1, CD97 andlatrophilin would be used in to determine their relative activities. Theresults can be applied accordingly. For example, a competitive bindingassay with PRO228 and CD97 can be performed with the ligand for CD97,CD55.

Native PRO533 is a 216 amino acid polypeptide of which residues 1-22 arethe signal sequence. Residues 3 to 216 have a Blast score of 509,corresponding to 53% homology to fibroblast growth factor. At thenucleotide level, DNA47412, the EST from which PCR oligos were generatedto isolate the full length DNA49435-1219, has been observed to map to11p15. Sequence homology to the 11p15 locus would indicate that PRO533may have utility in the treatment of Usher Syndrome or Atrophia areata.

As mentioned previously, fibroblast growth factors can act upon cells inboth a mitogenic and non-mitogenic manner. These factors are mitogenicfor a wide variety of normal diploid mesoderm-derived and neuralcrest-derived cells, inducing granulosa cells, adrenal cortical cells,chrondrocytes, myoblasts, corneal and vascular endothelial cells (bovineor human), vascular smooth muscle cells, lens, retina and prostaticepithelial cells, oligodendrocytes, astrocytes, chrondocytes, myoblastsand osteoblasts.

Non-mitogenic actions of fibroblast growth factors include promotion ofcell migration into a wound area (chemotaxis), initiation of new bloodvessel formulation (angiogenesis), modulation of nerve regeneration andsurvival (neurotrophism), modulation of endocrine functions, andstimulation or suppression of specific cellular protein expression,extracellular matrix production and cell survival. Baird, A. & Bohlen,P., Handbook of Exp. Phrmacol. 95(1): 369-418 (1990). These propertiesprovide a basis for using fibroblast growth factors in therapeuticapproaches to accelerate wound healing, nerve repair, collateral bloodvessel formation, and the like. For example, fibroblast growth factors,have been suggested to minimize myocardium damage in heart disease andsurgery (U.S. Pat. No. 4,378,437).

Since the PRO245 polypeptide and nucleic acid encoding it possesssequence homology to a transmembrane protein tyrosine kinase protein andits encoding nucleic acid, probes based upon the PRO245 nucleotidesequence may be employed to identify other novel transmembrane tyrosinekinase proteins. Soluble forms of the PRO245 polypeptide may be employedas antagonists of membrane bound PRO245 activity both in vitro and invivo. PRO245 polypeptides may be employed in screening assays designedto identify agonists or antagonists of the native PRO245 polypeptide,wherein such assays may take the form of any conventional cell-type orbiochemical binding assay. Moreover, the PRO245 polypeptide may serve asa molecular marker for the tissues in which the polypeptide isspecifically expressed.

PRO220, PRO221 and PRO227 all have leucine rich repeats. Additionally,PRO220 and PRO221 have homology to SLIT and leucine rich repeat protein.Therefore, these proteins are useful in assays described in theliterature, supra, wherein the SLIT and leucine rich repeat protein areused. Regarding the SLIT protein, PRO227 can be used in an assay todetermine the affect of PRO227 on neurodegenerative disease.Additionally, PRO227 has homology to human glycoprotein V. In the caseof PRO227, this polypeptide is used in an assay to determine its affecton bleeding, clotting, tissue repair and scarring.

The PRO266 polypeptide can be used in assays to determine if it has arole in neurodegenerative diseases or their reversal.

PRO269 polypeptides and portions thereof which effect the activity ofthrombin may also be useful for in vivo therapeutic purposes, as well asfor various in vitro applications. In addition, PRO269 polypeptides andportions thereof may have therapeutic use as an antithrombotic agentwith reduced risk for hemorrhage as compared with heparin. Peptideshaving homology to thrombomodulin are particularly desirable.

PRO287 polypeptides and portions thereof which effect the activity ofbone morphogenic protein “BMP1”/procollagen C-proteinase (PCP) may alsobe useful for in vivo therapeutic purposes, as well as for various invitro applications. In addition, PRO287 polypeptides and portionsthereof may have therapeutic applications in wound healing and tissuerepair. Peptides having homology to procollagen C-proteinase enhancerprotein and its precursor may also be used to induce bone and/orcartilage formation and are therefore of particular interest to thescientific and medical communities.

Therapeutic indications for PRO214 polypeptides include disordersassociated with the preservation and maintenance of gastrointestinalmucosa and the repair of acute and chronic mucosal lesions (e.g.,enterocolitis, Zollinger-Ellison syndrome, gastrointestinal ulcerationand congenital microvillus atrophy), skin diseases associated withabnormal keratinocyte differentiation (e.g., psoriasis, epithelialcancers such as lung squamous cell carcinoma, epidermoid carcinoma ofthe vulva and gliomas.

Studies on the generation and analysis of mice deficient in members ofthe TGF-superfamily are reported in Matzuk, Trends in Endocrinol. andMetabol., 6: 120-127 (1995).

The PRO317 polypeptide, as well as PRO317-specific antibodies,inhibitors, agonists, receptors, or their analogs, herein are useful intreating PRO317-associated disorders. Hence, for example, they may beemployed in modulating endometrial bleeding angiogenesis, and may alsohave an effect on kidney tissue. Endometrial bleeding can occur ingynecological diseases such as endometrial cancer as abnormal bleeding.Thus, the compositions herein may find use in diagnosing and treatingabnormal bleeding conditions in the endometrium, as by reducing oreliminating the need for a hysterectomy. The molecules herein may alsofind use in angiogenesis applications such as anti-tumor indications forwhich the antibody against vascular endothelial growth factor is used,or, conversely, ischemic indications for which vascular endothelialgrowth factor is employed.

Bioactive compositions comprising PRO317 or agonists or antagoniststhereof may be administered in a suitable therapeutic dose determined byany of several methodologies including clinical studies on mammalianspecies to determine maximal tolerable dose and on normal human subjectsto determine safe dose. Additionally, the bioactive agent may becomplexed with a variety of well established compounds or compositionswhich enhance stability or pharmacological properties such as half-life.It is contemplated that the therapeutic, bioactive composition may bedelivered by intravenous infusion into the bloodstream or any othereffective means which could be used for treating problems of the kidney,uterus, endometrium, blood vessels, or related tissue, e.g., in theheart or genital tract.

Dosages and administration of PRO317, PRO317 agonist, or PRO317antagonist in a pharmaceutical composition may be determined by one ofordinary skill in the art of clinical pharmacology or pharmacokinetics.See, for example, Mordenti and Rescigno, Pharmaceutical Research,9:17-25 (1992); Morenti et al., Pharmaceutical Research, 8:1351-1359(1991); and Mordenti and Chappell, “The use of interspecies scaling intoxicokinetics” in Toxicokinetics and New Drug Development, Yacobi etal. (eds) (Pergamon Press: NY, 1989), pp. 42-96. An effective amount ofPRO317, PRO317 agonist, or PRO317 antagonist to be employedtherapeutically will depend, for example, upon the therapeuticobjectives, the route of administration, and the condition of themammal. Accordingly, it will be necessary for the therapist to titer thedosage and modify the route of administration as required to obtain theoptimal therapeutic effect. A typical daily dosage might range fromabout 10 ng/kg to up to 100 mg/kg of the mammal's body weight or moreper day, preferably about 1 μg/kg/day to 10 mg/kg/day. Typically, theclinician will administer PRO317, PRO317 agonist, or PRO317 antagonist,until a dosage is reached that achieves the desired effect for treatmentof the above mentioned disorders.

PRO317 or an PRO317 agonist or PRO317 antagonist may be administeredalone or in combination with another to achieve the desiredpharmacological effect. PRO317 itself, or agonists or antagonists ofPRO317 can provide different effects when administered therapeutically.Such compounds for treatment will be formulated in a nontoxic, inert,pharmaceutically acceptable aqueous carrier medium preferably at a pH ofabout 5 to 8, more preferably 6 to 8, although the pH may vary accordingto the characteristics of the PRO317, agonist, or antagonist beingformulated and the condition to be treated. Characteristics of thetreatment compounds include solubility of the molecule, half-life, andantigenicity/immunogenicity; these and other characteristics may aid indefining an effective carrier.

PRO317 or PRO317 agonists or PRO317 antagonists may be delivered byknown routes of administration including but not limited to topicalcreams and gels; transmucosal spray and aerosol, transdermal patch andbandage; injectable, intravenous, and lavage formulations; and orallyadministered liquids and pills, particularly formulated to resiststomach acid and enzymes. The particular formulation, exact dosage, androute of administration will be determined by the attending physicianand will vary according to each specific situation.

Such determinations of administration are made by considering multiplevariables such as the condition to be treated, the type of mammal to betreated, the compound to be administered, and the pharmacokineticprofile of the particular treatment compound. Additional factors whichmay be taken into account include disease state (e.g. severity) of thepatient, age, weight, gender, diet, time of administration, drugcombination, reaction sensitivities, and tolerance/response to therapy.Long-acting treatment compound formulations (such as liposomallyencapsulated PRO317 or PEGylated PRO317 or PRO317 polymericmicrospheres, such as polylactic acid-based microspheres) might beadministered every 3 to 4 days, every week, or once every two weeksdepending on half-life and clearance rate of the particular treatmentcompound.

Normal dosage amounts may vary from about 10 ng/kg to up to 100 mg/kg ofmammal body weight or more per day, preferably about 1 μg/kg/day to 10mg/kg/day, depending upon the route of administration. Guidance as toparticular dosages and methods of delivery is provided in theliterature; see, for example, U.S. Pat. Nos. 4,657,760; 5,206,344; or5,225,212. It is anticipated that different formulations will beeffective for different treatment compounds and different disorders,that administration targeting the uterus, for example, may necessitatedelivery in a manner different from that to another organ or tissue,such as cardiac tissue.

Where sustained-release administration of PRO317 is desired in aformulation with release characteristics suitable for the treatment ofany disease or disorder requiring administration of PRO317,microencapsulation of PRO317 is contemplated. Microencapsulation ofrecombinant proteins for sustained release has been successfullyperformed with human growth hormone (rhGH), interferon-(rhIFN-),interleukin-2, and MN rgp120. Johnson et al., Nat. Med., 2: 795-799(1996); Yasuda. Biomed. Ther., 27: 1221-1223 (1993); Hora et al.,Bio/Technology, 8: 755-758 (1990); Cleland, “Design and Production ofSingle Immunization Vaccines Using Polylactide Polyglycolide MicrosphereSystems,” in Vaccine Design: The Subunit and Adjuvant Approach, Powelland Newman, eds, (Plenum Press: New York, 1995), pp. 439-462; WO97/03692, WO 96/40072, WO 96/07399; and U.S Pat. No. 5,654,010.

It is contemplated that conditions or diseases of the uterus,endometrial tissue, or other genital tissues or cardiac tissues mayprecipitate damage that is treatable with PRO317 or PRO317 agonist wherePRO317 expression is reduced in the diseased state; or with antibodiesto PRO317 or other PRO317 antagonists where the expression of PRO317 isincreased in the diseased state. These conditions or diseases may bespecifically diagnosed by the probing tests discussed above forphysiologic and pathologic problems which affect the function of theorgan.

The PRO317, PRO317 agonist, or PRO317 antagonist may be administered toa mammal with another biologically active agent, either separately or inthe same formulation to treat a common indication for which they areappropriate. For example, it is contemplated that PRO317 can beadministered together with EBAF-1 for those indications on which theydemonstrate the same qualitative biological effects. Alternatively,where they have opposite effects, EBAF-1 may be administered togetherwith an antagonist to PRO317, such as an anti-PRO317 antibody. Further,PRO317 may be administered together with VEGF for coronary ischemiawhere such indication is warranted, or with an anti-VEGF for cancer aswarranted, or, conversely, an antagonist to PRO317 may be administeredwith VEGF for coronary ischemia or with anti-VEGF to treat cancer aswarranted. These administrations would be in effective amounts fortreating such disorders.

Native PRO301 (SEQ ID NO:119) has a Blast score of 246 and 30% homologyat residues 24 to 282 of FIG. 44 with A33_HUMAN, an A33 antigenprecursor. A33 antigen precursor, as explained in the Background is atumor-specific antigen, and as such, is a recognized marker andtherapeutic target for the diagnosis and treatment of colon cancer. Theexpression of tumor-specific antigens is often associated with theprogression of neoplastic tissue disorders. Native PRO301 (SEQ IDNO:119) and A33_HUMAN also show a Blast score of 245 and 30% homology atresidues 21 to 282 of FIG. 44 with A33_HUMAN, the variation dependentupon how spaces are inserted into the compared sequences. Native PRO301(SEQ ID NO:119) also has a Blast score of 165 and 29% homology atresidues 60 to 255 of FIG. 44 with HS46KDA_(—)1, a human coxsackie andadenovirus receptor protein, also known as cell surface protein HCAR.This region of PRO301 also shows a similar Blast score and homology withHSU90716_(—)1. Expression of such proteins is usually associated withviral infection and therapeutics for the prevention of such infectionmay be accordingly conceived. As mentioned in the Background, theexpression of viral receptors is often associated with neoplastictumors.

Therapeutic uses for the PRO234 polypeptides of the invention includestreatments associated with leukocyte homing or the interaction betweenleukocytes and the endothelium during an inflammatory response. Examplesinclude asthma, rheumatoid arthritis, psoriasis and multiple sclerosis.

Since the PRO231 polypeptide and nucleic acid encoding it possesssequence homology to a putative acid phosphatase and its encodingnucleic acid, probes based upon the PRO231 nucleotide sequence may beemployed to identify other novel phosphatase proteins. Soluble forms ofthe PRO231 polypeptide may be employed as antagonists of membrane boundPRO231 activity both in vitro and in vivo. PRO231 polypeptides may beemployed in screening assays designed to identify agonists orantagonists of the native PRO231 polypeptide, wherein such assays maytake the form of any conventional cell-type or biochemical bindingassay. Moreover, the PRO231 polypeptide may serve as a molecular markerfor the tissues in which the polypeptide is specifically expressed.

PRO229 polypeptides can be fused with peptides of interest to determinewhether the fusion peptide has an increased half-life over the peptideof interest. The PRO229 polypeptides can be used accordingly to increasethe half-life of polypeptides of interest. Portions of PRO229 whichcause the increase in half-life are an embodiment of the inventionherein.

PRO238 can be used in assays which measure its ability to reducesubstrates, including oxygen and Aceyl-CoA, and particularly, measurePRO238's ability to produce oxygen free radicals. This is done by usingassays which have been previously described. PRO238 can further be usedto assay for candidates which block, reduce or reverse its reducingabilities. This is done by performing side by side assays wherecandidates are added in one assay having PRO238 and a substrate toreduce, and not added in another assay, being the same but for the lackof the presence of the candidate.

PRO233 polypeptides and portions thereof which have homology toreductase may also be useful for in vivo therapeutic purposes, as wellas for various other applications. The identification of novel reductaseproteins and related molecules may be relevant to a number of humandisorders such as inflammatory disease, organ failure, atherosclerosis,cardiac injury, infertility, birth defects, premature aging, AIDS,cancer, diabetic complications and mutations in general. Given thatoxygen free radicals and antioxidants appear to play important roles ina number of disease processes, the identification of new reductaseproteins and reductase-like molecules is of special importance in thatsuch proteins may serve as potential therapeutics for a variety ofdifferent human disorders. Such polypeptides may also play importantroles in biotechnological and medical research, as well as variousindustrial applications. As a result, there is particular scientific andmedical interest in new molecules, such as PRO233.

The PRO223 polypeptides of the present invention which exhibit serinecarboxypeptidease activity may be employed in vivo for therapeuticpurposes as well as for in vitro purposes. Those of ordinary skill inthe art will well know how to employ PRO223 polypeptides for such uses.

PRO235 polypeptides and portions thereof which may be involved in celladhesion are also useful for in vivo therapeutic purposes, as well asfor various in vitro applications. In addition, PRO235 polypeptides andportions thereof may have therapeutic applications in disease stateswhich involve cell adhesion. Given the physiological importance of celladhesion mechanisms in vivo, efforts are currently being under taken toidentify new, native proteins which are involved in cell adhesion.Therefore, peptides having homology to plexin are of particular interestto the scientific and medical communities.

Because the PRO236 and PRO262 polypeptides disclosed herein arehomologous to various known β-galactosidase proteins, the PRO236 andPRO262 polypeptides disclosed herein will find use in conjugates ofmonoclonal antibodies and the polypeptide for specific killing of tumorcells by generation of active drug from a galactosylated prodrug (e.g.,the generation of 5-fluorouridine from the prodrugβ-D-galactosyl-5-fluorouridine). The PRO236 and PRO262 polypeptidesdisclosed herein may also find various uses both in vivo and in vitro,wherein those uses will be similar or identical to uses for whichβ-galactosidase proteins are now employed. Those of ordinary skill inthe art will well know how to employ PRO236 and PRO262 polypeptides forsuch uses.

PRO239 polypeptides and portions thereof which have homology to densinmay also be useful for in vivo therapeutic purposes, as well as forvarious in vitro applications. In addition, PRO239 polypeptides andportions thereof may have therapeutic applications in disease stateswhich involve synaptic mechanisms, regeneration or cell adhesion. Giventhe physiological importance of synaptic processes, regeneration andcell adhesion mechanisms in vivo, efforts are currently being undertaken to identify new, native proteins which are involved in synapticmachinery and cell adhesion. Therefore, peptides having homology todensin are of particular interest to the scientific and medicalcommunities.

The PRO260 polypeptides described herein can be used in assays todetermine their relation to fucosidase. In particular, the PRO260polypeptides can be used in assays in determining their ability toremove fucose or other sugar residues from proteoglycans. The PRO260polypeptides can be assayed to determine if they have any functional orlocational similarities as fucosidase. The PRO260 polypeptides can thenbe used to regulate the systems in which they are integral.

PRO263 can be used in assays wherein CD44 antigen is generally used todetermine PRO263 activity relative to that of CD44. The results can beused accordingly.

PRO270 polypeptides and portions thereof which effectreduction-oxidation (redox) state may also be useful for in vivotherapeutic purposes, as well as for various in vitro applications. Morespecifically, PRO270 polypeptides may affect the expression of a largevariety of genes thought to be involved in the pathogenesis of AIDS,cancer, atherosclerosis, diabetic complications and in pathologicalconditions involving oxidative stress such as stroke and inflammation.In addition, PRO270 polypeptides and portions thereof may affect theexpression of a genes which have a role in apoptosis. Therefore,peptides having homology to thioredoxin are particularly desirable tothe scientific and medical communities.

PRO272 polypeptides and portions thereof which possess the ability tobind calcium may also have numerous in vivo therapeutic uses, as well asvarious in vitro applications. Therefore, peptides having homology toreticulocalbin are particularly desirable. Those with ordinary skill inthe art will know how to employ PRO272 polypeptides and portions thereoffor such purposes.

PRO294 polypeptides and portions thereof which have homology to collagenmay also be useful for in vivo therapeutic purposes, as well as forvarious other applications. The identification of novel collagens andcollage-like molecules may have relevance to a number of humandisorders. Thus, the identification of new collagens and collage-likemolecules is of special importance in that such proteins may serve aspotential therapeutics for a variety of different human disorders. Suchpolypeptides may also play important roles in biotechnological andmedical research as well as various industrial applications. Given thelarge number of uses for collagen, there is substantial interest inpolypeptides with homology to the collagen molecule.

PRO295 polypeptides and portions thereof which have homology to integrinmay also be useful for in vivo therapeutic purposes, as well as forvarious other applications. The identification of novel integrins andintegrin-like molecules may have relevance to a number of humandisorders such as modulating the binding or activity of cells of theimmune system. Thus, the identification of new integrins andintegrin-like molecules is of special importance in that such proteinsmay serve as potential therapeutics for a variety of different humandisorders. Such polypeptides may also play important roles inbiotechnological and medical research as well as various industrialapplications. As a result, there is particular scientific and medicalinterest in new molecules, such as PRO295.

As the PRO293 polypeptide is clearly a leucine rich repeat polypeptidehomologue, the peptide can be used in all applications that the knownNLRR-1 and NLRR-2 polypeptides are used. The activity can be comparedbetween these peptides and thus applied accordingly.

The PRO247 polypeptides described herein can be used in assays in whichdensin is used to determine the activity of PRO247 relative to densin orthese other proteins. The results can be used accordingly in diagnosticsand/or therapeutic applications with PRO247.

PRO302, PRO303, PRO304, PRO307 and PRO343 polypeptides of the presentinvention which possess protease activity may be employed both in vivofor therapeutic purposes and in vitro. Those of ordinary skill in theart will well know how to employ the PRO302, PRO303, PRO304, PRO307 andPRO343 polypeptides of the present invention for such purposes.

PRO328 polypeptides and portions thereof which have homology to GLIP andCRISP may also be useful for in vivo therapeutic purposes, as well asfor various other applications. The identification of novel GLIP andCRISP-like molecules may have relevance to a number of human disorderswhich involve transcriptional regulation or are over expressed in humantumors. Thus, the identification of new GLIP and CRISP-like molecules isof special importance in that such proteins may serve as potentialtherapeutics for a variety of different human disorders. Suchpolypeptides may also play important roles in biotechnological andmedical research as well as in various industrial applications. As aresult, there is particular scientific and medical interest in newmolecules, such as PRO328.

Uses for PRO335, PRO331 or PRO326 including uses in competitive assayswith LIG-1, ALS and decorin to determine their relative activities. Theresults can be used accordingly. PRO335, PRO331 or PRO326 can also beused in assays where LIG-1 would be used to determine if the sameeffects are incurred.

PRO332 contains GAG repeat (GKEK) at amino acidpositions 625-628 in FIG.108 (SEQ ID NO:310). Slippage in such repeats can be associated withhuman disease. Accordingly, PRO332 can use useful for the treatment ofsuch disease conditions by gene therapy, i.e. by introduction of a genecontaining the correct GKEK sequence motif.

Other uses of PRO334 include use in assays in which fibrillin or fibulinwould be used to determine the relative activity of PRO334 to fibrillinor fibulin. In particular, PRO334 can be used in assays which requirethe mechanisms imparted by epidermal growth factor repeats.

Native PRO346 (SEQ ID NO:320) has a Blast score of 230, corresponding to27% homology between amino acid residues 21 to 343 with residues 35 to1040 CGM6_HUMAN, a carcinoembryonic antigen cgm6 precursor. Thishomology region includes nearly all but 2 N-terminal extracellulardomain residues, including an immunoglobulin superfamily homology atresidues 148 to 339 of PRO346 in addition to several transmembraneresidues (340-343). Carcinoembryonic antigen precursor, as explained inthe Background is a tumor-specific antigen, and as such, is a recognizedmarker and therapeutic target for the diagnosis and treatment of coloncancer. The expression of tumor-specific antigens is often associatedwith the progression of neoplastic tissue disorders. Native PRO346 (SEQID NO:320) and P_W06874, a human carcinoembryonic antigen CEA-d have aBlast score of 224 and homology of 28% between residues 2 to 343 and 67to 342, respectively. This homology includes the entire extracellulardomain residues of native PRO346, minus the initiator methionine(residues 2 to 18) as well as several transmembrane residues (340-343).

PRO268 polypeptides which have protein disulfide isomerase activity willbe useful for many applications where protein disulfide isomeraseactivity is desirable including, for example, for use in promotingproper disulfide bond formation in recombinantly produced proteins so asto increase the yield of correctly folded protein. Those of ordinaryskill in the art will readily know how to employ such PRO268polypeptides for such purposes.

PRO330 polypeptides of the present invention which possess biologicalactivity related to that of the prolyl 4-hydroxylase alpha subunitprotein may be employed both in vivo for therapeutic purposes and invitro. Those of ordinary skill in the art will well know how to employthe PRO330 polypeptides of the present invention for such purposes.

Uses of the herein disclosed molecules may also be based upon thepositive functional assay hits disclosed and described below.

F. Anti-PRO Antibodies

The present invention further provides anti-PRO antibodies. Exemplaryantibodies include polyclonal, monoclonal, humanized, bispecific, andheteroconjugate antibodies.

-   -   1. Polyclonal Antibodies

The anti-PRO antibodies may comprise polyclonal antibodies. Methods ofpreparing polyclonal antibodies are known to the skilled artisan.Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Theimmunizing agent may include the PRO polypeptide or a fusion proteinthereof. It may be useful to conjugate the immunizing agent to a proteinknown to be immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Examples of adjuvants which may be employed include Freund'scomplete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). The immunization protocol may beselected by one skilled in the art without undue experimentation.

-   -   2. Monoclonal Antibodies

The anti-PRO antibodies may, alternatively, be monoclonal antibodies.Monoclonal antibodies may be prepared using hybridoma methods, such asthose described by Kohler and Milstein, Nature 256:495 (1975). In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes may beimmunized in vitro.

The immunizing agent will typically include the PRO polypeptide or afusion protein thereof. Generally, either peripheral blood lymphocytes(“PBLs”) are used if cells of human origin are desired, or spleen cellsor lymph node cells are used if non-human mammalian sources are desired.The lymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell [Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103]. Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine and human origin. Usually, rat or mouse myeloma cell lines areemployed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001(1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against PRO.Preferably, the binding specificity of monoclonal antibodies produced bythe hybridoma cells is determined by immunoprecipitation or by an invitro binding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). Such techniques and assays are known inthe art. The binding affinity of the monoclonal antibody can, forexample, be determined by the Scatchard analysis of Munson and Pollard,Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods[Goding, supra]. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences [U.S. Pat.No. 4,816,567; Morrison et al., supra] or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart.

-   -   3. Human and Humanized Antibodies

The anti-PRO antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introducing of human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992);Lonberg et al., Nature 368 856-859(1994); Morrison, Nature 368, 812-13(1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995).

-   -   4. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forthe PRO, the other one is for any other antigen, and preferably for acell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities [Milsteinand Cuello, Nature, 305:537-539 (1983)]. Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO 96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the CH3region of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniques forgenerating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared can be prepared using chemical linkage. Brennan et al., Science229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Fab′ fragments may be directly recovered from E. coli and chemicallycoupled to form bispecific antibodies. Shalaby et al., J. Exp. Med.175:217-225 (1992) describe the production of a fully humanizedbispecific antibody F(ab′)₂ molecule. Each Fab′ fragment was separatelysecreted from E. coli and subjected to directed chemical coupling invitro to form the bispecific antibody. The bispecific antibody thusformed was able to bind to cells overexpressing the ErbB2 receptor andnormal human T cells, as well as trigger the lytic activity of humancytotoxic lymphocytes against human breast tumor targets.

Various technique for making and isolating bispecific antibody fragmentsdirectly from recombinant cell culture have also been described. Forexample, bispecific antibodies have been produced using leucine zippers.Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipperpeptides from the Fos and Jun proteins were linked to the Fab′ portionsof two different antibodies by gene fusion. The antibody homodimers werereduced at the hinge region to form monomers and then re-oxidized toform the antibody heterodimers. This method can also be utilized for theproduction of antibody homodimers. The “diabody” technology described byHollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) hasprovided an alternative mechanism for making bispecific antibodyfragments. The fragments comprise a heavy-chain variable domain (V_(H))connected to a light-chain variable domain (V_(L)) by a linker which istoo short to allow pairing between the two domains on the same chain.Accordingly, the V_(H) and V_(L) domains of one fragment are forced topair with the complementary V_(L) and V_(H) domains of another fragment,thereby forming two antigen-binding sites. Another strategy for makingbispecific antibody fragments by the use of single-chain Fv (sFv) dimershas also been reported. See, Gruber et al., J. Immunol. 152:5368 (1994).Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

Exemplary bispecific antibodies may bind to two different epitopes on agiven PRO polypeptide herein. Alternatively, an anti-PRO polypeptide armmay be combined with an arm which binds to a triggering molecule on aleukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32)and FcγRIII (CD16) so as to focus cellular defense mechanisms to thecell expressing the particular PRO polypeptide. Bispecific antibodiesmay also be used to localize cytotoxic agents to cells which express aparticular PRO polypeptide. These antibodies possess a PRO-binding armand an arm which binds a cytotoxic agent or a radionuclide chelator,such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody ofinterest binds the PRO polypeptide and further binds tissue factor (TF).

-   -   5. Heteroconiugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of twocovalentlyjoined antibodies. Such antibodies have, for example, beenproposed to target immune system cells to unwanted cells [U.S. Pat. No.4,676,980], and for treatment of HIV infection [WO 91/00360; WO92/200373; EP 03089]. It is contemplated that the antibodies may beprepared in vitro using known methods in synthetic protein chemistry,including those involving crosslinking agents. For example, immunotoxinsmay be constructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

-   -   6. Effector Function Engineering

It may be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating cancer. For example, cysteine residue(s) may beintroduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedmay have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195(1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimericantibodies with enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. CancerResearch, 53: 2560-2565 (1993). Alternatively, an antibody can beengineered that has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design, 3: 219-230 (1989).

-   -   7. Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

In another embodiment, the antibody may be conjugated to a “receptor”(such streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g., avidin) that is conjugatedto a cytotoxic agent (e.g., a radionucleotide).

-   -   8. Immunoliposomes

The antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al., J. National Cancer Inst.,81(19): 1484 (1989).

-   -   9. Pharmaceutical Compositions of Antibodies

Antibodies specifically binding a PRO polypeptide identified herein, aswell as other molecules identified by the screening assays disclosedhereinbefore, can be administered for the treatment of various disordersin the form of pharmaceutical compositions.

If the PRO polypeptide is intracellular and whole antibodies are used asinhibitors, internalizing antibodies are preferred. However,lipofections or liposomes can also be used to deliver the antibody, oran antibody fragment, into cells. Where antibody fragments are used, thesmallest inhibitory fragment that specifically binds to the bindingdomain of the target protein is preferred. For example, based upon thevariable-region sequences of an antibody, peptide molecules can bedesigned that retain the ability to bind the target protein sequence.Such peptides can be synthesized chemically and/or produced byrecombinant DNA technology. See, e.g., Marasco et al., Proc. Natl. Acad.Sci. USA, 90: 7889-7893 (1993). The formulation herein may also containmore than one active compound as necessary for the particular indicationbeing treated, preferably those with complementary activities that donot adversely affect each other. Alternatively, or in addition, thecomposition may comprise an agent that enhances its function, such as,for example, a cytotoxic agent, cytokine, chemotherapeutic agent, orgrowth-inhibitory agent. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS-S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

G. Uses for Anti-PRO Antibodies

The anti-PRO antibodies of the invention have various utilities. Forexample, anti-PRO antibodies may be used in diagnostic assays for PRO,e.g., detecting its expression in specific cells, tissues, or serum.Various diagnostic assay techniques known in the art may be used, suchas competitive binding assays, direct or indirect sandwich assays andimmunoprecipitation assays conducted in either heterogeneous orhomogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques,CRC Press, Inc. (1987) pp. 147-158]. The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase. Any method known in theart for conjugating the antibody to the detectable moiety may beemployed, including those methods described by Hunter et al., Nature,144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al.,J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. andCytochem., 30:407 (1982).

Anti-PRO antibodies also are useful for the affinity purification of PROfrom recombinant cell culture or natural sources. In this process, theantibodies against PRO are immobilized on a suitable support, such aSephadex resin or filter paper, using methods well known in the art. Theimmobilized antibody then is contacted with a sample containing the PROto be purified, and thereafter the support is washed with a suitablesolvent that will remove substantially all the material in the sampleexcept the PRO, which is bound to the immobilized antibody. Finally, thesupport is washed with another suitable solvent that will release thePRO from the antibody.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Rockville, Md.

Example 1 Extracellular Domain Homology Screening to Identify NovelPolypeptides and cDNA Encoding Therefor

The extracellular domain (ECD) sequences (including the secretion signalsequence, if any) from about 950 known secreted proteins from theSwiss-Prot public database were used to search EST databases. The ESTdatabases included public databases (e.g., Dayhoff, GenBank), andproprietary databases (e.g. LIFESEQ™, Incyte Pharmaceuticals, Palo Alto,Calif.). The search was performed using the computer program BLAST orBLAST2 (Altschul, and Gish, Methods in Enzymology 266: 460-80 (1996); asa comparison of the ECD protein sequences to a 6 frame translation ofthe EST sequences. Those comparisons with a Blast score of 70 (or insome cases 90) or greater that did not encode known proteins wereclustered and assembled into consensus DNA sequences with the program“phrap” (Phil Green, University of Washington, Seattle, Wash.).

Using this extracellular domain homology screen, consensus DNA sequenceswere assembled relative to the other identified EST sequences. Inaddition, the consensus DNA sequences obtained were often (but notalways) extended using repeated cycles of BLAST and phrap to extend theconsensus sequence as far as possible using the sources of EST sequencesdiscussed above.

Based upon the consensus sequences obtained as described above,oligonucleotides were then synthesized and used to identify by PCR acDNA library that contained the sequence of interest and for use asprobes to isolate a clone of the full-length coding sequence for a PROpolypeptide. Forward (.f) and reverse (.r) PCR primers generally rangefrom 20 to 30 nucleotides and are often designed to give a PCR productof about 100-1000 bp in length. The probe (.p) sequences are typically40-55 bp in length. In some cases, additional oligonucleotides aresynthesized when the consensus sequence is greater than about 1-0.5 kbp.In order to screen several libraries for a full-length clone, DNA fromthe libraries was screened by PCR amplification, as per Ausubel et al.,Current Protocols in Molecular Biology, with the PCR primer pair. Apositive library was then used to isolate clones encoding the gene ofinterest using the probe oligonucleotide and one of the primer pairs.

The cDNA libraries used to isolate the cDNA clones were constructed bystandard methods using commercially available reagents such as thosefrom Invitrogen, San Diego, Calif. The cDNA was primed with oligo dTcontaining a NotI site, linked with blunt to SalI hemikinased adaptors,cleaved with NotI, sized appropriately by gel electrophoresis, andcloned in a defined orientation into a suitable cloning vector (such aspRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain theSfiI site; see, Holmes et al., Science, 253:1278-1280 (1991)) in theunique XhoI and NotI sites.

Example 2 Isolation of cDNA Clones Encoding PRO211 and PRO217

Consensus DNA sequences were assembled as described in Example 1 aboveand were designated as DNA28730 and DNA28760, respectively. Based onthese consensus sequences, oligonucleotides were synthesized and used toidentify by PCR a cDNA library that contained the sequences of interestand for use as probes to isolate a clone of the full-length codingsequence for the PRO211 and PRO217 polypeptides. The libraries used toisolate DNA32292-1131 and DNA33094-1131 were fetal lung libraries.

cDNA clones were sequenced in their entirety. The entire nucleotidesequences of PRO211 (DNA32292-1131) and PRO217 (UNQ191) are shown inFIG. 1 (SEQ ID NO:1) and FIG. 3 (SEQ ID NO:3), respectively. Thepredicted polypeptides are 353 and 379 amino acid in length,respectively, with respective molecular weights of approximately 38,190and 41,520 daltons.

The oligonucleotide sequences used in the above procedures were thefollowing:

28730.p (OLI 516) (SEQ ID NO:5)5′-AGGGAGCACGGACAGTGTGCAGATGTGGACGAGTGCTCACTAGCA-3′ 28730.f (OLI 517)(SEQ ID NO:6) 5′-AGAGTGTATCTCTGGCTACGC-3′ 28730.r (OLI 518) (SEQ IDNO:7) 5′-TAAGTCCGGCACATTACAGGTC-3′ 28760.p (OLI 617) (SEQ ID NO:8)5′-CCCACGATGTATGAATGGTGGACTTTGTGTGACTCCTGGTTTCTGCATC-3′ 28760.f (OLI618) (SEQ ID NO:9) 5′-AAAGACGCATCTGCGAGTGTCC-3′ 28760.r (OLI 619) (SEQID NO:10) 5′-TGCTGATTTCACACTGCTCTCCC-3′

Example 3 Isolation of cDNA Clones Encoding Human PRO230

A consensus DNA sequence was assembled relative to the other identifiedEST sequences as described in Example 1 above, wherein the consensussequence is designated herein as DNA30857. An EST proprietary toGenentech was employed in the consensus assembly. The EST is designatedas DNA20088 and has the nucleotide sequence shown in FIG. 7 (SEQ IDNO:13).

Based on the DNA30857 consensus sequence, oligonucleotides weresynthesized to identify by PCR a cDNA library that contained thesequence of interest and for use as probes to isolate a clone of thefull-length coding sequence for PRO230.

A pair of PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-TTCGAGGCCTCTGAGAAGTGGCCC-3′ (SEQ ID NO:14) reversePCR primer 5′-GGCGGTATCTCTCTGGCCTCCC-3′ (SEQ ID NO:15)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA30857 sequence which had the followingnucleotide sequencehybridization probe5′-TTCTCCACAGCAGCTGTGGCATCCGATCGTGTCTCAATCCATTCTCTGGG-3′   (SEQ IDNO:16)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO230 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetallung tissue. DNA sequencing of the clones isolated as described abovegave the full-length DNA sequence for PRO230 (herein designated asDNA33223-1136 and the derived protein sequence for PRO230.

The entire nucleotide sequence of DNA33223-1136 is shown in FIG. 5 (SEQID NO:11). Clone DNA33223-1136 contains a single open reading frame withan apparent translational initiation site at nucleotide positions100-103 and ending at the stop codon at nucleotide positions 1501-1503(FIG. 5; SEQ ID NO:11). The predicted polypeptide precursor is 467 aminoacids long FIG. 6).

Example 4 Isolation of cDNA Clones Encoding Human PRO232

A consensus DNA sequence was assembled relative to the other identifiedEST sequences as described in Example 1 above, wherein the consensussequence is designated herein as DNA30935. Based on the DNA30935consensus sequence, oligonucleotides were synthesized to identify by PCRa cDNA library that contained the sequence of interest and for use asprobes to isolate a clone of the full-length coding sequence for PRO232.

A pair of PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-TGCTGTGCTACTCCTGCAAAGCCC-3′ (SEQ ID NO:19) reversePCR primer 5′-TGCACAAGTCGGTGTCACAGCACG-3′ (SEQ ID NO:20)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA30935 sequence which had the followingnucleotide sequencehybridization probe5′-AGCAACGAGGACTGCCTGCAGGTGGAGAACTGCACCCAGCTGGG-3′   (SEQ ID NO:16)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO232 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalkidney tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO232 [herein designated as DNA34435-1140]and the derived protein sequence for PRO232.

The entire nucleotide sequence of DNA34435-1140 is shown in FIG. 8 (SEQID NO:17). Clone DNA34435-1140 contains a single open reading frame withan apparent translational initiation site at nucleotide positions 17-19and ending at the stop codon at nucleotide positions 359-361 (FIG. 8;SEQ ID NO:17). The predicted polypeptide precursor is 114 amino acidslong (FIG. 9). Clone DNA34435-1140 has been deposited with ATCC on Sep.16, 1997 and is assigned ATCC deposit no. ATCC 209250.

Analysis of the amino acid sequence of the full-length PRO232 suggeststhat it possesses 35% sequence identity with a stem cell surface antigenfrom Gallus gallus.

Example 5 Isolation of cDNA Clones Encoding PRO187

A proprietary expressed sequence tag (EST) DNA database (LIFESEQ™,Incyte Pharmaceuticals, Palo Alto, Calif.) was searched and an EST(#843193) was identified which showed homology to fibroblast growthfactor (FGF-8) also known as androgen-induced growth factor. mRNA wasisolated from human fetal lung tissue using reagents and protocols fromInvitrogen, San Diego, Calif. (Fast Track 2). The cDNA libraries used toisolate the cDNA clones were constructed by standard methods usingcommercially available reagents (e.g., Invitrogen, San Diego, Calif.,Life Technologies, Gaithersburg, Md.). The cDNA was primed with oligo dTcontaining a NotI site, linked with blunt to SalI hemikinased adaptors,cleaved with NotI, sized appropriately by gel electrophoresis, andcloned in a defined orientation into the cloning vector pRK5D usingreagents and protocols from Life Technologies, Gaithersburg, Md. (SuperScript Plasmid System). The double-stranded cDNA was sized to greaterthan 1000 bp and the SalI/NotI linkered cDNA was cloned into XhoI/NotIcleaved vector. pRK5D is a cloning vector that has an sp6 transcriptioninitiation site followed by an SfiI restriction enzyme site precedingthe XhoI/NotI cDNA cloning sites.

Several libraries from various tissue sources were screened by PCRamplification with the following oligonucleotide probes:

IN843193.f (OLI315) (SEQ ID NO:24) 5′-CAGTACGTGAGGGACCAGGGCGCCATGA-3′IN843193.r (OLI 317) (SEQ ID NO:25) 5′-CCGGTGACCTGCACGTGCTTGCCA-3′A positive library was then used to isolate clones encoding the PRO187gene using one of the above oligonucleotides and the followingoligonucleotide probe:IN843193. p (OLI 316)   (SEQ ID NO:26)5′-GCGGATCTGCCGCCTGCTCANCTGGTCGGTCATGGCGCCCT-3′

A cDNA clone was sequenced in entirety. The entire nucleotide sequenceof PRO187 (DNA27864-1155) is shown in FIG. 10 (SEQ ID NO:22). CloneDNA27864-1155 contains a single open reading frame with an apparenttranslational initiation site at nucleotide position 1 (FIG. 10; SEQ IDNO:22). The predicted polypeptide precursor is 205 amino acids long.Clone DNA27864-1155 has been deposited with the ATCC (designation:DNA27864-1155) and is assigned ATCC deposit no. ATCC 209375.

Based on a BLAST and FastA sequence alignment analysis (using the ALIGNcomputer program) of the full-length sequence, the PRO187 polypeptideshows 74% amino acid sequence identity (Blast score 310) to humanfibroblast growth factor-8 (androgen-induced growth factor).

Example 6 Isolation of cDNA Clones Encoding PRO265

A consensus DNA sequence was assembled relative to other EST sequencesas described in Example 1 above using phrap. This consensus sequence isherein designated DNA33679. Based on the DNA33679 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO265.

PCR primers (two forward and one reverse) were synthesized:

forward PCR primer A: 5′-CGGTCTACCTGTATGGCAACC-3′ (SEQ ID NO:29);forward PCR primer B: 5′-GCAGGACAACCAGATAAACCAC-3′ (SEQ ID NO:30);reverse PCR primer 5′-ACGCAGATTTGAGAAGGCTGTC-3′ (SEQ ID NO:31)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA33679 sequence which had the followingnucleotide sequencehybridization probe5′-TTCACGGGCTGCTCTTGCCCAGCTCTTGAAGCTTGAAGAGCTGCAC-3′   (SEQ ID NO:32)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with PCRprimer pairs identified above. A positive library was then used toisolate clones encoding the PRO265 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human afetal brain library.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO265 [herein designated as DNA36350-1158](SEQ ID NO:27) and the derived protein sequence for PRO265.

The entire nucleotide sequence of DNA36350-1158 is shown in FIG. 12 (SEQID NO:27). Clone DNA36350-1158 contains a single open reading frame withan apparent translational initiation site at nucleotide positions352-354 and ending at the stop codon at positions 2332-2334 (FIG. 12).The predicted polypeptide precursor is 660 amino acids long (FIG. 13).Clone DNA36350-1158 has been deposited with ATCC and is assigned ATCCdeposit no. ATCC 209378.

Analysis of the amino acid sequence of the full-length PRO265polypeptide suggests that portions of it possess significant homology tothe fibromodulin and the fibromodulin precursor, thereby indicating thatPRO265 may be a novel member of the leucine rich repeat family,particularly related to fibromodulin.

Example 7 Isolation of cDNA Clones Encoding Human PRO219

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA28729. Based on the DNA28729 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO219.

A pair of PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-GTGACCCTGGTTGTGAATACTCC-3′ (SEQ ID NO:35) reversePCR primer 5′-ACAGCCATGGTCTATAGCTTGG-3′ (SEQ ID NO:36)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA28729 sequence which had the followingnucleotide sequencehybridization probe5′-GCCTGTCAGTGTCCTGAGGGACACGTGCTCCGCAGCGATGGGAAG-3′   (SEQ ID NO:37)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO219 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalkidney tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO219 [herein designated as DNA32290-1164](SEQ ID NO:33) and the derived protein sequence for PRO219.

The entire nucleotide sequence of DNA32290-1164 is shown in FIG. 14 (SEQID NO:33). Clone DNA32290-1164 contains a single open reading frame withan apparent translational initiation site at nucleotide positions204-206 and ending at the stop codon at nucleotide positions 2949-2951(FIG. 14). The predicted polypeptide precursor is 915 amino acids long(FIG. 15). Clone DNA32290-1164 has been deposited with ATCC and isassigned ATCC deposit no. ATCC 209384.

Analysis of the amino acid sequence of the full-length PRO219polypeptide suggests that portions of it possess significant homology tothe mouse and human matrilin-2 precursor polypeptides.

Example 8 Isolation of cDNA Clones Encoding Human PRO246

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA30955. Based on the DNA30955 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO246.

A pair of PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-AGGGTCTCCAGGAGAAAGACTC-3′ (SEQ ID NO:40) reversePCR primer 5′-ATTGTGGGCCTTGCAGACATAGAC-3′ (SEQ ID NO:41)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA30955 sequence which had the followingnucleotide sequencehybridization probe5′-GGCCACAGCATCAAAACCTTAGAACTCAATGTACTGGTTCCTCCAGCTCC-3′   (SEQ IDNO:42)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO246 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalliver tissue. DNA sequencing of the clones isolated as described abovegave the full-length DNA sequence for PRO246 [herein designated asDNA35639-1172] (SEQ ID NO:38) and the derived protein sequence forPRO246.

The entire nucleotide sequence of DNA35639-1172 is shown in FIG. 16 (SEQID NO:38). Clone DNA35639-1172 contains a single open reading frame withan apparent translational initiation site at nucleotide positions126-128 and ending at the stop codon at nucleotide positions 1296-1298(FIG. 16). The predicted polypeptide precursor is 390 amino acids long(FIG. 17). Clone DNA35639-1172 has been deposited with ATCC and isassigned ATCC deposit no. ATCC 209396.

Analysis of the amino acid sequence of the full-length PRO246polypeptide suggests that it possess significant homology to the humancell surface protein HCAR, thereby indicating that PRO246 may be a novelcell surface virus receptor.

Example 9 Isolation of cDNA Clones Encoding Human PRO228

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA28758. An EST proprietary to Genentech was employedin the consensus assembly. This EST is shown in FIG. 20 (SEQ ID NO:50)and is herein designated as DNA21951.

Based on the DNA28758 consensus sequence, oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence for PRO228.

PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-GGTAATGAGCTCCATTACAG-3′ (SEQ ID NO:51) forward PCRprimer 5′-GGAGTAGAAAGCGCATGG-3′ (SEQ ID NO:52) forward PCR primer5′-CACCTGATACCATGAATGGCAG-3′ (SEQ ID NO:53) reverse PCR primer5′-CGAGCTCGAATTAATTCG-3′ (SEQ ID NO:54) reverse PCR primer5′-GGATCTCCTGAGCTCAGG-3′ (SEQ ID NO:55) reverse PCR primer5′-CCTAGTTGAGTGATCCTTGTAAG-3′ (SEQ ID NO:56)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA28758 sequence which had the followingnucleotide sequencehybridization probe5′-ATGAGACCCACACCTCATGCCGCTGTAATCACCTGACACATTTTGCAATT-3′   (SEQ IDNO:57)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pairs identified above. A positive library was then used toisolate clones encoding the PRO228 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalkidney tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO228 [herein designated as DNA33092-1202](SEQ ID NO:48) and the derived protein sequence for PRO228.

The entire nucleotide sequence of DNA33092-1202 is shown in FIG. 18 (SEQID NO:48). Clone DNA33092-1202 contains a single open reading frame withan apparent translational initiation site at nucleotide positions 24-26of SEQ ID NO:48 and ending at the stop codon after nucleotide position2093 of SEQ ID NO:48. The predicted polypeptide precursor is 690 aminoacids long (FIG. 19). Clone DNA33092-1202 has been deposited with ATCCand is assigned ATCC deposit no. ATCC 209420.

Analysis of the amino acid sequence of the full-length PRO228polypeptide suggests that portions of it possess significant homology tothe secretin-related proteins CD97 and EMR1 as well as the secretinmember, latrophilin, thereby indicating that PRO228 may be a new memberof the secretin related proteins.

Example 10 Isolation of cDNA Clones Encoding Human PRO533

The EST sequence accession number AF007268, a murine fibroblast growthfactor (FGF-15) was used to search various public EST databases (e.g.,GenBank, Dayhoff, etc.). The search was performed using the computerprogram BLAST or BLAST2 [Altschul et al., Methods in Enzymology,266:460-480 (1996)] as a comparison of the ECD protein sequences to a 6frame translation of the EST sequences. The search resulted in a hitwith GenBank EST AA220994, which has been identified as stratagene NT2neuronal precursor 937230.

Based on the Genbank EST AA220994 sequence, oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence. Forward and reverse PCR primers may rangefrom 20 to 30 nucleotides (typically about 24), and are designed to givea PCR product of 100-1000 bp in length. The probe sequences aretypically 40-55 bp (typically about 50) in length. In order to screenseveral libraries for a source of a full-length clone, DNA from thelibraries was screened by PCR amplification, as per Ausubel et al.,Current Protocols in Molecular Biology, with the PCR primer pair. Apositive library was then used to isolate clones encoding the gene ofinterest using the probe oligonucleotide and one of the PCR primers.

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified below. A positive library was then used toisolate clones encoding the PRO533 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalretina. The cDNA libraries used to isolated the cDNA clones wereconstructed by standard methods using commercially available reagents(e.g., Invitrogen, San Diego, Calif.; Clontech, etc.) The cDNA wasprimed with oligo dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

A cDNA clone was sequenced in its entirety. The full length nucleotidesequence of PRO533 is shown in FIG. 21 (SEQ ID NO:58). CloneDNA49435-1219 contains a single open reading frame with an apparenttranslational initiation site at nucleotide positions 459461 (FIG. 21;SEQ ID NO:58). The predicted polypeptide precursor is 216 amino acidslong. Clone DNA47412-1219 has been deposited with ATCC and is assignedATCC deposit no. ATCC 209480.

Based on a BLAST-2 and FastA sequence alignment analysis of thefull-length sequence, PRO533 shows amino acid sequence identity tofibroblast growth factor (53%). The oligonucleotide sequences used inthe above procedure were the following:

FGF15.forward: 5′-ATCCGCCCAGATGGCTACAATGTGTA-3′ (SEQ ID NO:60);FGF15.probe: 5′-GCCTCCCGGTCTCCCTGAGCAGTGCCAAACAGCGGCAGTGTA-3′ (SEQ IDNO:61); FGF15.reverse: 5′-CCAGTCCGGTGACAAGCCCAAA-3′ (SEQ ID NO:62).

Example 11 Isolation of cDNA Clones Encoding Human PRO245

A consensus DNA sequence was assembled relative to the other identifiedEST sequences as described in Example 1 above, wherein the consensussequence is designated herein as DNA30954.

Based on the DNA30954 consensus sequence, oligonucleotides weresynthesized to identify by PCR a cDNA library that contained thesequence of interest and for use as probes to isolate a clone of thefull-length coding sequence for PRO245.

A pair of PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-ATCGTTGTGAAGTTAGTGCCCC-3′ (SEQ ID NO:65) reversePCR primer 5′-ACCTGCGATATCCAACAGAATTG-3′ (SEQ ID NO:66)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA30954 sequence which had the followingnucleotide sequencehybridization probe5′-GGAAGAGGATACAGTCACTCTGGAAGTATTAGTGGCTCCAGCAGTTCC-3′   (SEQ ID NO:67)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO245 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalliver tissue. DNA sequencing of the clones isolated as described abovegave the full-length DNA sequence for PRO245 [herein designated asDNA35638-1141] and the derived protein sequence for PRO245.

The entire nucleotide sequence of DNA35638-1141 is shown in FIG. 23 (SEQID NO:63). Clone DNA35638-1141 contains a single open reading frame withan apparent translational initiation site at nucleotide positions 89-91and ending at the stop codon at nucleotide positions 1025-1027 (FIG. 23;SEQ ID NO:63). The predicted polypeptide precursor is 312 amino acidslong (FIG. 24). Clone DNA35638-1141 has been deposited with ATCC on Sep.16, 1997 and is assigned ATCC deposit no. ATCC 209265.

Analysis of the amino acid sequence of the full-length PRO245 suggeststhat a portion of it possesses 60% amino acid identity with the humanc-myb protein and, therefore, may be a new member of the transmembraneprotein receptor tyrosine kinase family.

Example 12 Isolation of cDNA Clones Encoding Human PRO220, PRO221 andPRO227

(a) PRO220

A consensus DNA sequence was assembled relative to the other identifiedEST sequences as described in Example 1 above, wherein the consensussequence is designated herein as DNA28749. Based on the DNA28749consensus sequence, oligonucleotides were synthesized to identify by PCRa cDNA library that contained the sequence of interest and for use asprobes to isolate a clone of the full-length coding sequence for PRO220.

A pair of PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-TCACCTGGAGCCTTTATTGGCC-3′ (SEQ ID NO:74) reversePCR primer 5′-ATACCAGCTATAACCAGGCTGCG-3′ (SEQ ID NO:75)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA28749 sequence which had the followingnucleotide sequence:hybridization probe5′-CAACAGTAAGTGGTTTGATGCTCTTCCAAATCTAGAGATTCTGATGATTGGG-3′   (SEQ IDNO:76).

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO220 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetallung tissue. DNA sequencing of the clones isolated as described abovegave the full-length DNA sequence for PRO220 [herein designated asDNA32298-1132 and the derived protein sequence for PRO220.

The entire nucleotide sequence of DNA32298-1132 is shown in FIG. 25 (SEQID NO:68). Clone DNA32298-1132 contains a single open reading frame withan apparent translational initiation site at nucleotide positions480-482 and ending at the stop codon at nucleotide positions 2604-2606(FIG. 25). The predicted polypeptide precursor is 708 amino acids long(FIG. 26). Clone DNA32298-1132 has been deposited with ATCC and isassigned ATCC deposit no. ATCC 209257.

Analysis of the amino acid sequence of the full-length PRO220 shows ithas homology to member of the leucine rich repeat protein superfamily,including the leucine rich repeat protein and the neuronal leucine-richrepeat protein 1.

(b) PRO221

A consensus DNA sequence was assembled relative to the other identifiedEST sequences as described in Example 1 above, wherein the consensussequence is designated herein as DNA28756. Based on the DNA28756consensus sequence, oligonucleotides were synthesized to identify by PCRa cDNA library that contained the sequence of interest and for use asprobes to isolate a clone of the full-length coding sequence for PRO221.

A pair of PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-CCATGTGTCTCCTCCTACAAAG-3′ (SEQ ID NO:77) reversePCR primer 5′-GGGAATAGATGTGATCTGATTGG-3′ (SEQ ID NO:78)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA28756 sequence which had the followingnucleotide sequence:hybridization probe5′-CACCTGTAGCAATGCAAATCTCAAGGAAATACCTAGAGATCTTCCTCCTG-3′   (SEQ IDNO:79)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO221 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetallung tissue. DNA sequencing of the clones isolated as described abovegave the full-length DNA sequence for PRO221 [herein designated asDNA33089-1132 and the derived protein sequence for PRO221.

The entire nucleotide sequence of DNA33089-1132 is shown in FIG. 27 (SEQID NO:70). Clone DNA33089-1132 contains a single open reading frame withan apparent translational initiation site at nucleotide positions179-181 and ending at the stop codon at nucleotide positions 956-958(FIG. 27). The predicted polypeptide precursor is 259 amino acids long(FIG. 28). PRO221 is believed to have a transmembrane region at aminoacids 206-225. Clone DNA33089-1132 has been deposited with ATCC and isassigned ATCC deposit no. ATCC 209262.

Analysis of the amino acid sequence of the full-length PRO221 shows ithas homology to member of the leucine rich repeat protein superfamily,including the SLIT protein.

(c) PRO227

A consensus DNA sequence was assembled relative to the other identifiedEST sequences as described in Example 1 above, wherein the consensussequence is designated herein as DNA28740. Based on the DNA28740consensus sequence, oligonucleotides were synthesized to identify by PCRa cDNA library that contained the sequence of interest and for use asprobes to isolate a clone of the full-length coding sequence for PRO227.

A pair of PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-AGCAACCGCCTGAAGCTCATCC-3′ (SEQ ID NO:80) reversePCR primer 5′-AAGGCGCGGTGAAAGATGTAGACG-3′ (SEQ ID NO:81)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA28740 sequence which had the followingnucleotide sequence:hybridization probe5′GACTACATGTTTCAGGACCTGTACAACCTCAAGTCACTGGAGGTTGGCGA-3′   (SEQ IDNO:82).

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO227 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetallung tissue. DNA sequencing of the clones isolated as described abovegave the full-length DNA sequence for PRO227 [herein designated asDNA33786-1132 and the derived protein sequence for PRO227.

The entire nucleotide sequence of DNA33786-1132 is shown in FIG. 29 (SEQID NO:72). Clone DNA33786-1132 contains a single open reading frame withan apparent translational initiation site at nucleotide positions 33-35and ending at the stop codon at nucleotide positions 1893-1895 (FIG.29). The predicted polypeptide precursor is 620 amino acids long (FIG.30). PRO227 is believed to have a transmembrane region. CloneDNA33786-1132 has been deposited with ATCC and is assigned ATCC depositno. ATCC 209253.

Analysis of the amino acid sequence of the full-length PRO221 shows ithas homology to member of the leucine rich repeat protein superfamily,including the platelet glycoprotein V precursor and the humanglycoprotein V.

Example 13 Isolation of cDNA Clones Encoding Human PRO258

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA28746.

Based on the DNA28746 consensus sequence, oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence for PRO258.

PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-GCTAGGAATTCCACAGAAGCCC-3′ (SEQ ID NO:85) reversePCR primer 5′-AACCTGGAATGTCACCGAGCTG-3′ (SEQ ID NO:86) reverse PCRprimer 5′-CCTAGCACAGTGACGAGGGACTTGGC-3′ (SEQ ID NO:87)Additionally, synthetic oligonucleotide hybridization probes wereconstructed from the consensus DNA28740 sequence which had the followingnucleotide sequence:

hybridization probe5′-AAGACACAGCCACCCTAAACTGTCAGTCTTCTGGGAGCAAGCCTGCAGCC-3′ (SEQ ID NO:88)5′-GCCCTGGCAGACGAGGGCGAGTACACCTGCTCAATCTTCACTATGCCTGT-3′ (SEQ ID NO:89)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO258 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetallung tissue. DNA sequencing of the clones isolated as described abovegave the full-length DNA sequence for PRO258 [herein designated asDNA35918-1174] (SEQ ID NO:83) and the derived protein sequence forPRO258.

The entire nucleotide sequence of DNA35918-1174 is shown in FIG. 31 (SEQID NO:83). Clone DNA35918-1174 contains a single open reading frame withan apparent translational initiation site at nucleotide positions147-149 of SEQ ID NO:83 and ending at the stop codon after nucleotideposition 1340 of SEQ ID NO:83 (FIG. 31). The predicted polypeptideprecursor is 398 amino acids long (FIG. 32). Clone DNA35918-1174 hasbeen deposited with ATCC and is assigned ATCC deposit no. ATCC 209402.

Analysis of the amino acid sequence of the full-length PRO258polypeptide suggests that portions of it possess significant homology tothe CRTAM and the poliovirus receptor and have an Ig domain, therebyindicating that PRO258 is a new member of the Ig superfamily.

Example 14 Isolation of cDNA Clones Encoding Human PRO266

An expressed sequence tag database was searched for ESTs having homologyto SLIT, resulting in the identification of a single EST sequencedesignated herein as T73996. Based on the T73996 EST sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO266.

A pair of PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-GTTGGATCTGGGCAACAATAAC-3′ (SEQ ID NO:92) reversePCR primer 5′-ATTGTTGTGCAGGCTGAGTTTAAG-3′ (SEQ ID NO:93)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed which had the following nucleotide sequencehybridization probe5′-GGTGGCTATACATGGATAGCAATTACCTGGACACGCTGTCCCGGG-3′   (SEQ ID NO:94)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO266 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalbrain tissue. DNA sequencing of the clones isolated as described abovegave the full-length DNA sequence for PRO266 [herein designated asDNA37150-1178] (SEQ ID NO:90) and the derived protein sequence forPRO266.

The entire nucleotide sequence of DNA37150-1178 is shown in FIG. 33 (SEQID NO:90). Clone DNA37150-1178 contains a single open reading frame withan apparent translational initiation site at nucleotide positions167-169 and ending at the stop codon after nucleotide position 2254 ofSEQ ID NO:90. The predicted polypeptide precursor is 696 amino acidslong (FIG. 34). Clone DNA37150-1178 has been deposited with ATCC and isassigned ATCC deposit no. ATCC 209401.

Analysis of the amino acid sequence of the full-length PRO266polypeptide suggests that portions of it possess significant homology tothe SLIT protein, thereby indicating that PRO266 may be a novel leucinerich repeat protein.

Example 15 Isolation of cDNA Clones Encoding Human PRO269

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA35705. Based on the DNA35705 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO269.

Forward and reverse PCR primers were synthesized:

forward PCR primer (.f1) 5′-TGGAAGGAGATGCGATGCCACCTG-3′ (SEQ ID NO:97)forward PCR primer (.f2) 5′-TGACCAGTGGGGAAGGACAG-3′ (SEQ ID NO:98)forward PCR primer (.f3) 5′-ACAGAGCAGAGGGTGCCTTG-3′ (SEQ ID NO:99)reverse PCR primer (.r1) 5′-TCAGGGACAAGTGGTGTCTCTCCC-3′ (SEQ ID NO:100)reverse PCR primer (.r2) 5′-TCAGGGAAGGAGTGTGCAGTTCTG-3′ (SEQ ID NO:101)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA35705 sequence which had the followingnucleotide sequence:hybridization probe5′-ACAGCTCCCGATCTCAGTTACTTGCATCGCGGACGAAATCGGCGCTCGCT-3′   (SEQ IDNO:102)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pairs identified above. A positive library was then used toisolate clones encoding the PRO269 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalkidney tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO269 [herein designated as DNA38260-1180](SEQ ID NO:95) and the derived protein sequence for PRO269.

The entire nucleotide sequence of DNA38260-1180 is shown in FIG. 35 (SEQID NO:95). Clone DNA38260-1180 contains a single open reading frame withan apparent translational initiation site at nucleotide positions314-316 and ending at the stop codon at nucleotide positions 1784-1786(FIG. 35; SEQ ID NO:95). The predicted polypeptide precursor is 490amino acids long (FIG. 36). Clone DNA38260-1180 has been deposited withATCC and is assigned ATCC deposit no. ATCC 209397.

Analysis of the amino acid sequence of the full-length PRO269 suggeststhat portions of it possess significant homology to the humanthrombomodulin proteins, thereby indicating that PRO269 may possess oneor more thrombomodulin-like domains.

Example 16 Isolation of cDNA Clones Encoding Human PRO287

A consensus DNA sequence encoding PRO287 was assembled relative to theother identified EST sequences as described in Example 1 above, whereinthe consensus sequence is designated herein as DNA28728. Based on theDNA28728 consensus sequence, oligonucleotides were synthesized toidentify by PCR a cDNA library that contained the sequence of interestand for use as probes to isolate a clone of the full-length codingsequence for PRO287.

A pair of PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-CCGATTCATAGACCTCGAGAGT-3′ (SEQ ID NO:105) reversePCR primer 5′-GTCAAGGAGTCCTCCACAATAC-3′ (SEQ ID NO:106)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA28728 sequence which had the followingnucleotide sequencehybridization probe5′-GTGTACAATGGCCATGCCAATGGCCAGCGCATTGGCCGCTTCTGT-3′ (SEQ ID NO:107)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO287 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalkidney tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO287 [herein designated as DNA39969-1185,SEQ ID NO:103] and the derived protein sequence for PRO287.

The entire nucleotide sequence of DNA39969-1185 is shown in FIG. 37 (SEQID NO:103). Clone DNA39969-1185 contains a single open reading framewith an apparent translational initiation site at nucleotide positions307-309 and ending at the stop codon at nucleotide positions 1552-1554(FIG. 37; SEQ ID NO:103). The predicted polypeptide precursor is 415amino acids long (FIG. 38). Clone DNA39969-1185 has been deposited withATCC and is assigned ATCC deposit no. ATCC 209400.

Analysis of the amino acid sequence of the full-length PRO287 suggeststhat it may possess one or more procollagen C-proteinase enhancerprotein precursor or procollagen C-proteinase enhancer protein-likedomains. Based on a BLAST and FastA sequence alignment analysis of thefull-length sequence, PRO287 shows nucleic acid sequence identity toprocollagen C-proteinase enhancer protein precursor and procollagenC-proteinase enhancer protein (47 and 54%, respectively).

Example 17 Isolation of cDNA Clones Encoding Human PRO214

A consensus DNA sequence was assembled using phrap as described inExample 1 above. This consensus DNA sequence is designated herein asDNA28744. Based on this consensus sequence, oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence.

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified below. A positive library was then used toisolate clones encoding the PRO214 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetallung tissue.

A cDNA clone was sequenced in its entirety The full length nucleotidesequence of DNA32286-1191 is shown in FIG. 39 (SEQ ID NO:108).DNA32286-1191 contains a single open reading frame with an apparenttranslational initiation site at nucleotide position 103 (FIG. 39; SEQID NO:108). The predicted polypeptide precursor is 420 amino acids long(SEQ ID NO:109).

Based on a BLAST and FastA sequence alignment analysis of thefull-length sequence, PRO214 polypeptide shows amino acid sequenceidentity to HT protein and/or Fibulin (49% and 38%, respectively).

The oligonucleotide sequences used in the above procedure were thefollowing:

28744.p (OLI555)5′-CCTGGCTATCAGCAGGTGGGCTCCAAGTGTCTCGATGTGGATGAGTGTGA-3′ (SEQ ID NO:110)28744.f (OLI556) 5′-ATTCTGCGTGAACACTGAGGGC-3′ (SEQ ID NO:111) 28744.r(OLI557) 5′-ATCTGCTTGTAGCCCTCGGCAC-3′ (SEQ ID NO:112)

Example 18 Isolation of cDNA Clones Encoding Human PRO317

A consensus DNA sequence was assembled using phrap as described inExample 1 above, wherein the consensus sequence is herein designated asDNA28722. Based on this consensus sequence, oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence. The forward and reverse PCR primers,respectively, synthesized for this purpose were:

5′-AGGACTGCCATAACTTGCCTG (OLI489) (SEQ ID NO:115) and5′-ATAGGAGTTGAAGCAGCGCTGC (OLI490) (SEQ ID NO:116).

The probe synthesized for this purpose was:5′-TGTGTGGACATAGACGAGTGCCGCTACCGCTACTGCCAGCACCGC (OL1488) (SEQ IDNO:117)

mRNA for construction of the cDNA libraries was isolated from humanfetal kidney tissue.

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification, as perAusubel et al., Current Protocols in Molecular Biology (1989), with thePCR primer pair identified above. A positive library was then used toisolate clones containing the PRO317 gene using the probeoligonucleotide identified above and one of the PCR primers.

A cDNA clone was sequenced in its entirety. The entire nucleotidesequence of DNA33461-1199 (encoding PRO317) is shown in FIG. 41 (SEQ IDNO:113). Clone DNA33461-1199 contains a single open reading frame withan apparent translational initiation site at nucleotide positions 68-70(FIG. 41; SEQ ID NO:113). The predicted polypeptide precursor is 366amino acids long. The predicted signal sequence is amino acids 1-18 ofFIG. 42 (SEQ ID NO:114). There is one predicted N-linked glycosylationsite at amino acid residue 160. Clone DNA33461-1199 has been depositedwith ATCC and is assigned ATCC deposit no. ATCC 209367.

Based on BLAST™ and FastA™ sequence alignment analysis (using the ALIGN™computer program) of the full-length PRO317 sequence, PRO317 shows themost amino acid sequence identity to EBAF-1 (92%). The results alsodemonstrate a significant homology between human PRO317 and mouse LEFTYprotein. The C-terminal end of the PRO317 protein contains manyconserved sequences consistent with the pattern expected of a member ofthe TGF-superfamily.

In situ expression analysis in human tissues performed as describedbelow evidences that there is distinctly strong expression of the PRO317polypeptide in pancreatic tissue.

Example 19 Isolation of cDNA clones Encoding Human PRO301

A consensus DNA sequence designated herein as DNA35936 was assembledusing phrap as described in Example 1 above. Based on this consensussequence, oligonucleotides were synthesized: 1) to identify by PCR acDNA library that contained the sequence of interest, and 2) for use asprobes to isolate a clone of the full-length coding sequence.

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified below. A positive library was then used toisolate clones encoding the PRO301 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalkidney.

A cDNA clone was sequenced in its entirety. The full length nucleotidesequence of native sequence PRO301 is shown in FIG. 43 (SEQ ID NO:118).Clone DNA40628-1216 contains a single open reading frame with anapparent translational initiation site at nucleotide positions 52-54(FIG. 43; SEQ ID NO:118). The predicted polypeptide precursor is 299amino acids long with a predicted molecular weight of 32,583 daltons andpI of 8.29. Clone DNA40628-1216 has been deposited with ATCC and isassigned ATCC deposit No. ATCC 209432.

Based on a BLAST and FastA sequence alignment analysis of thefull-length sequence, PRO301 shows amino acid sequence identity to A33antigen precursor (30%) and coxsackie and adenovirus receptor protein(29%).

The oligonucleotide sequences used in the above procedure were thefollowing:

OLI2162 (35936.f1) 5′-TCGCGGAGCTGTGTTCTGTTTCCC-3′ (SEQ ID NO:120)OLI2163 (35936.p1)5′-TGATCGCGATGGGGACAAAGGCGCAAGCTCGAGAGGAAACTGTTGTGCCT-3′ (SEQ ID NO:121)OLI2164 (35936.f2) 5′-ACACCTGGTTCAAAGATGGG-3′ (SEQ ID NO:122) OLI2165(35936.r1) 5′-TAGGAAGAGTTGCTGAAGGCACGG-3′ (SEQ ID NO:123) OLI2166(35936.f3) 5′-TTGCCTTACTCAGGTGCTAC-3′ (SEQ ID NO:124) OLI2167 (35936.r2)5′-ACTCAGCAGTGGTAGGAAAG-3′ (SEQ ID NO:125)

Example 20 Isolation of cDNA Clones Encoding Human PRO224

A consensus DNA sequence assembled relative to the other identified ESTsequences as described in Example 1, wherein the consensus sequence isdesignated herein as DNA30845. Based on the DNA30845 consensus sequence,oligonucleotides were synthesized to identify by PCR a cDNA library thatcontained the sequence of interest and for use as probes to isolate aclone of the full-length coding sequence for PRO224.

A pair of PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-AAGTTCCAGTGCCGCACCAGTGGC-3′ (SEQ ID NO:128)reverse PCR primer 5′-TTGGTTCCACAGCCGAGCTCGTCG-3′ (SEQ ID NO:129)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA30845 sequence which had the followingnucleotide sequencehybridization probe5′-GAGGAGGAGTGCAGGATTGAGCCATGTACCCAGAAAGGGCAATGCCCACC-3′   (SEQ IDNO:130)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO224 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalliver tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO224 [herein designated as DNA33221-1133]and the derived protein sequence for PRO224.

The entire nucleotide sequence of DNA33221-1133 is shown in FIG. 45 (SEQID NO:126). Clone DNA33221-1133 contains a single open reading framewith an apparent translational initiation site at nucleotide positions33-35 and ending at the stop codon at nucleotide positions 879-899 (FIG.45; SEQ ID NO:126). The start of a transmembrane region begins atnucleotide position 777. The predicted polypeptide precursor is 282amino acids long (FIG. 46). Clone DNA33221-1133 has been deposited withATCC and is assigned ATCC deposit no. ATCC 209263.

Analysis of the amino acid sequence of the full-length PRO224 suggeststhat it has homology to very low-density lipoprotein receptors,apolipoprotein E receptor and chicken oocyte receptors P95. Based on aBLAST and FastA sequence alignment analysis of the full-length sequence,PRO224 has amino acid identity to portions of these proteins in therange from 28% to 45%, and overall identity with these proteins in therange from 33% to 39%.

Example 21 Isolation of cDNA Clones Encoding Human PRO222

A consensus DNA sequence was assembled relative to the other identifiedEST sequences as described in Example 1 above, wherein the consensussequence is designated herein as DNA28771. Based on the DNA28771consensus sequence, oligonucleotides were synthesized to identify by PCRa cDNA library that contained the sequence of interest and for use asprobes to isolate a clone of the full-length coding sequence for PRO222.

A pair of PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-ATCTCCTATCGCTGCTTTCCCGG-3′ (SEQ ID NO:133) reversePCR primer 5′-AGCCAGGATCGCAGTAAAACTCC-3′ (SEQ ID NO:134)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA28771 sequence which had the followingnucleotide sequence:hybridization probe5′-ATTTAAACTTGATGGGTCTGCGTATCTTGAGTGCTTACAAAACCTTATCT-3′   (SEQ IDNO:135)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO222 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalkidney tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO222 [herein designated as DNA33107-1135]and the derived protein sequence for PRO222.

The entire nucleotide sequence of DNA33107-1135 is shown in FIG. 47 (SEQID NO:131). Clone DNA33107-1135 contains a single open reading framewith an apparent translational initiation site at nucleotide positions159-161 and ending at the stop codon at nucleotide positions 1629-1631(FIG. 47; SEQ ID NO:131). The predicted polypeptide precursor is 490amino acids long (FIG. 48). Clone DNA33107-1135 has been deposited withATCC and is assigned ATCC deposit no. ATCC 209251.

Based on a BLAST and FastA sequence alignment analysis of thefull-length sequence, PRO222 shows amino acid sequence identity to mousecomplement factor h precursor (25-26%), complement receptor (27-29%),mouse complement C3b receptor type 2 long form precursor (2547%) andhuman hypothetical protein kiaa0247 (40%).

Example 22 Isolation of cDNA clones Encoding PRO234

A consensus DNA sequence was assembled (DNA30926) using phrap asdescribed in Example 1 above. Based on this consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence.

RNA for the construction of the cDNA libraries was isolated usingstandard isolation protocols, e.g., Ausubel et al., Current Protocols inMolecular Biology, from tissue or cell line sources or it was purchasedfrom commercial sources (e.g., Clontech). The cDNA libraries used toisolate the cDNA clones were constructed by standard methods (e.g.,Ausubel et al.) using commercially available reagents (e.g.,Invitrogen). This library was derived from 22 week old fetal braintissue.

A cDNA clone was sequenced in its entirety. The entire nucleotidesequence of PRO234 is shown in FIG. 49 (SEQ ID NO:136). The predictedpolypeptide precursor is 382 amino acids long and has a calculatedmolecular weight of approximately 43.1 kDa.

The oligonucleotide sequences used in the above procedure were thefollowing:

30926.p (OLI826) (SEQ ID NO:138): 5′-GTTCATTGAAAACCTCTTGCCATCTGATGGTGACTTCTGGATTGGGCTCA-3′ 30926.f (OLI827) (SEQ ID NO:139):5′-AAGCCAAAGAAGCCTGCAGGAGGG-3′ 30926.r (OLI828) (SEQ ID NO:140):5′-CAGTCCAAGCATAAAGGTCCTGGC-3′

Example 23 Isolation of cDNA Clones Encoding Human PRO231

A consensus DNA sequence was assembled relative to the other identifiedEST sequences as described in Example 1 above, wherein the consensussequence was designated herein as DNA30933. Based on the DNA30933consensus sequence, oligonucleotides were synthesized to identify by PCRa cDNA library that contained the sequence of interest and for use asprobes to isolate a clone of the full-length coding sequence for PRO231.

Three PCR primers (two forward and one reverse) were synthesized:

forward PCR primer 1 5′-CCAACTACCAAAGCTGCTGGAGCC-3′ (SEQ ID NO:143)forward PCR primer 2 5′-GCAGCTCTATTACCACGGGAAGGA-3′ (SEQ ID NO:144)reverse PCR primer 5′-TCCTTCCCGTGGTAATAGAGCTGC-3′ (SEQ ID NO:145)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA30933 sequence which had the followingnucleotide sequencehybridization probe5′-GGCAGAGAACCAGAGGCCGGAGGAGACTGCCTCTTTACAGCCAGG-3′   (SEQ ID NO:146)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pairs identified above. A positive library was then used toisolate clones encoding the PRO231 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalliver tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO231 [herein designated as DNA34434-1139]and the derived protein sequence for PRO231.

The entire nucleotide sequence of DNA34434-1139 is shown in FIG. 51 (SEQID NO:141). Clone DNA34434-1139 contains a single open reading framewith an apparent translational initiation site at nucleotide positions173-175 and ending at the stop codon at nucleotide positions 1457-1459(FIG. 51; SEQ ID NO:141). The predicted polypeptide precursor is 428amino acids long (FIG. 52). Clone DNA34434-1139 has been deposited withATCC on Sep. 16, 1997 and is assigned ATCC deposit no. ATCC 209252.

Analysis of the amino acid sequence of the full-length PRO231 suggeststhat it possesses 30% and 31% amino acid identity with the human and ratprostatic acid phosphatase precursor proteins, respectively.

Example 24 Isolation of cDNA Clones Encoding Human PRO229

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA28762. Based on the DNA28762 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO229.

A pair of PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-TTCAGCTCATCACCTTCACCTGCC-3′ (SEQ ID NO:149)reverse PCR primer 5′-GGCTCATACAAAATACCACTAGGG-3′ (SEQ ID NO:150)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA28762 sequence which had the followingnucleotide sequencehybridization probe5′-GGGCCTCCACCGCTGTGAAGGGCGGGTGGAGGTGGAACAGAAAGGCCAGT-3′   (SEQ IDNO:151)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO229 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalliver tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO229 [herein designated as DNA33100-1159](SEQ ID NO:147) and the derived protein sequence for PRO229.

The entire nucleotide sequence of DNA33100-1159 is shown in FIG. 53 (SEQID NO:147). Clone DNA33100-1159 contains a single open reading framewith an apparent translational initiation site at nucleotide positions98-100 and ending at the stop codon at nucleotide positions 1139-1141(FIG. 53). The predicted polypeptide precursor is 347 amino acids long(FIG. 54). Clone DNA33100-1159 has been deposited with ATCC and isassigned ATCC deposit no.ATCC 209377.

Analysis of the amino acid sequence of the full-length PRO229polypeptide suggests that portions of it possess significant homology toantigen wc1.1, M130 antigen and CD6.

Example 25 Isolation of cDNA Clones Encoding Human PRO238

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described above in Example 1. This consensus sequence isherein designated DNA30908. Based on the DNA30908 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO238.

PCR primers (forward and reverse) were synthesized:

forward PCR primer 1 5′-GGTGCTAAACTGGTGCTCTGTGGC-3′ (SEQ ID NO:154)forward PCR primer 2 5′-CAGGGCAAGATGAGCATTCC-3′ (SEQ ID NO:155) reversePCR primer 5′-TCATACTGTTCCATCTCGGCACGC-3′ (SEQ ID NO:156)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA30908 sequence which had the followingnucleotide sequencehybridization probe5′-AATGGTGGGGCCCTAGAAGAGCTCATCAGAGAACTCACCGCTTCTCATGC-3′   (SEQ IDNO:157)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO238 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalliver tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO238 and the derived protein sequence forPRO238.

The entire nucleotide sequence of DNA35600-1162 is shown in FIG. 55 (SEQID NO:152). Clone DNA35600-1162 contains a single open reading framewith an apparent translational initiation site at nucleotide positions134-136 and ending prior to the stop codon at nucleotide positions1064-1066 (FIG. 55). The predicted polypeptide precursor is 310 aminoacids long (FIG. 56). Clone DNA35600-1162 has been deposited with ATCCand is assigned ATCC deposit no. ATCC 209370.

Analysis of the amino acid sequence of the full-length PRO238polypeptide suggests that portions of it possess significant homology toreductase, particularly oxidoreductase, thereby indicating that PRO238may be a novel reductase.

Example 26 Isolation of cDNA Clones Encoding Human PRO233

The extracellular domain (ECD) sequences (including the secretionsignal, if any) of from about 950 known secreted proteins from theSwiss-Prot public protein database were used to search expressedsequence tag (EST) databases. The EST databases included public ESTdatabases (e.g., GenBank) and a proprietary EST DNA database (LIFESEQ™,Incyte Pharmaceuticals, Palo Alto, Calif.). The search was performedusing the computer program BLAST or BLAST2 (Altshul et al., Methods inEnzymology 266:460-480 (1996)) as a comparison of the ECD proteinsequences to a 6 frame translation of the EST sequence. Thosecomparisons resulting in a BLAST score of 70 (or in some cases 90) orgreater that did not encode known proteins were clustered and assembledinto consensus DNA sequences with the program “phrap” (Phil Green,University of Washington, Seattle, Wash.).

An expressed sequence tag (EST) was identified by the EST databasesearch and a consensus DNA sequence was assembled relative to other ESTsequences using phrap. This consensus sequence is herein designatedDNA30945. Based on the DNA30945 consensus sequence, oligonucleotideswere synthesized: 1) to identify by PCR a cDNA library that containedthe sequence of interest, and 2) for use as probes to isolate a clone ofthe full-length coding sequence for PRO233.

Forward and reverse PCR primers were synthesized:

forward PCR primer 5′-GGTGAAGGCAGAAATTGGAGATG-3′ (SEQ ID NO:160) reversePCR primer 5′-ATCCCATGCATCAGCCTGTTTACC-3′ (SEQ ID NO:161)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA30945 sequence which had the followingnucleotide sequencehybridization probe5′-GCTGGTGTAGTCTATACATCAGATTTGTTTGCTACACAAGATCCTCAG-3′ (SEQ ID NO:162)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO233 gene using the probe oligonucleotide.

RNA for construction of the cDNA libraries was isolated from human fetalbrain tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO233 [herein designated as DNA34436-1238](SEQ ID NO:158) and the derived protein sequence for PRO233.

The entire nucleotide sequence of DNA34436-1238 is shown in FIG. 57 (SEQID NO:158). Clone DNA34436-1238 contains a single open reading framewith an apparent translational initiation site at nucleotide positions101-103 and ending at the stop codon at nucleotide positions 1001-1003(FIG. 57). The predicted polypeptide precursor is 300 amino acids long(FIG. 58). The full-length PRO233 protein shown in FIG. 58 has anestimated molecular weight of about 32,964 daltons and a p1 of about9.52. Clone DNA34436-1238 has been deposited with ATCC and is assignedATCC deposit no. ATCC 209523.

Analysis of the amino acid sequence of the full-length PRO233polypeptide suggests that portions of it possess significant homology toreductase proteins, thereby indicating that PRO233 may be a novelreductase.

Example 27 Isolation of cDNA Clones Encoding Human PRO223

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA30836. Based on the DNA30836 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO223.

PCR primer pairs (one forward and two reverse) were synthesized:

forward PCR primer 5′-TTCCATGCCACCTAAGGGAGACTC-3′ (SEQ ID NO:165)reverse PCR primer 1 5′-TGGATGAGGTGTGCAATGGCTGGC-3′ (SEQ ID NO:166)reverse PCR primer 2 5′-AGCTCTCAGAGGCTGGTCATAGGG-3′ (SEQ ID NO:167)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA30836 sequence which had the followingnucleotide sequencehybridization probe5′-GTCGGCCCTTTCCCAGGACTGAACATGAAGAGTTATGCCGGCTTCCTCAC-3 ′   (SEQ IDNO:168)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO223 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalliver tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO223 [herein designated as DNA33206-1165](SEQ ID NO:163) and the derived protein sequence for PRO223.

The entire nucleotide sequence of DNA33206-1165 is shown in FIG. 59 (SEQID NO:163). Clone DNA33206-1165 contains a single open reading framewith an apparent translational initiation site at nucleotide positions97-99 and ending at the stop codon at nucleotide positions 1525-1527(FIG. 59). The predicted polypeptide precursor is 476 amino acids long(FIG. 60). Clone DNA33206-1165 has been deposited with ATCC and isassigned ATCC deposit no. ATCC 209372.

Analysis of the amino acid sequence of the full-length PRO223polypeptide suggests that it possesses significant homology to variousserine carboxypeptidase proteins, thereby indicating that PRO223 may bea novel serine carboxypeptidase.

Example 28 Isolation of cDNA Clones Encoding Human PRO235

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated “DNA30927”. Based on the DNA30927 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO235.

A pair of PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-TGGAATACCGCCTCCTGCAG-3′ (SEQ ID NO:171) reversePCR primer 5′-CTTCTGCCCTTTGGAGAAGATGGC-3′ (SEQ ID NO:172)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA30927 sequence which had the followingnucleotide sequencehybridization probe5′-GGACTCACTGGCCCAGGCCTTCAATATCACCAGCCAGGACGAT-3′   (SEQ ID NO:173)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO235 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalliver tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO235 [herein designated as DNA35558-1167](SEQ ID NO:169) and the derived protein sequence for PRO235.

The entire nucleotide sequence of DNA35558-1167 is shown in FIG. 61 (SEQID NO:169). Clone DNA35558-1167 contains a single open reading framewith an apparent translational initiation site at nucleotide positions667-669 and ending at the stop codon at nucleotide positions 2323-2325(FIG. 61). The predicted polypeptide precursor is 552 amino acids long(FIG. 62). Clone DNA35558-1167 has been deposited with ATCC and isassigned ATCC deposit no. 209374.

Analysis of the amino acid sequence of the full-length PRO235polypeptide suggests that portions of it possess significant homology tothe human, mouse and Xenopus plexin protein, thereby indicating thatPRO235 may be a novel plexin protein.

Example 29 Isolation of cDNA Clones Encoding Human PRO236 and HumanPRO262

Consensus DNA sequences were assembled relative to other EST sequencesusing phrap as described in Example 1 above. These consensus sequencesare herein designated DNA30901 and DNA30847. Based on the DNA30901 andDNA30847 consensus sequences, oligonucleotides were synthesized: 1) toidentify by PCR a cDNA library that contained the sequence of interest,and 2) for use as probes to isolate a clone of the full-length codingsequence for PRO236 and PRO262, respectively.

Based upon the DNA30901 consensus sequence, a pair of PCR primers(forward and reverse) were synthesized:

forward PCR primer 5′-TGGCTACTCCAAGACCCTGGCATG-3′ (SEQ ID NO:178)reverse PCR primer 5′-TGGACAAATCCCCTTGCTCAGCCC-3′ (SEQ ID NO:179)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA30901 sequence which had the followingnucleotide sequencehybridization probe5′-GGGCTTCACCGAAGCAGTGGACCTTTATTTTGACCACCTGATGTCCAGGG-3′   (SEQ IDNO:180)

Based upon the DNA30847 consensus sequence, a pair of PCR primers(forward and reverse) were synthesized:

forward PCR primer 5′-CCAGCTATGACTATGATGCACC-3′ (SEQ ID NO:181) reversePCR primer 5′-TGGCACCCAGAATGGTGTTGGCTC-3′ (SEQ ID NO:182)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA30847 sequence which had the followingnucleotide sequencehybridization probe5′-CGAGATGTCATCAGCAAGTTCCAGGAAGTTCCTTTGGGACCTTTACCTCC-3′   (SEQ IDNO:183)

In order to screen several libraries for a source of full-length clones,DNA from the libraries was screened by PCR amplification with the PCRprimer pairs identified above. Positive libraries were then used toisolate clones encoding the PRO236 and PRO262 genes using the probeoligonucleotides and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetallung tissue for PRO236 and human fetal liver tissue for PRO262.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO236 [herein designated as DNA35599-1168](SEQ ID NO:174), the derived protein sequence for PRO236, thefull-length DNA sequence for PRO262 [herein designated as DNA36992-1168](SEQ ID NO:176) and the derived protein sequence for PRO262.

The entire nucleotide sequence of DNA35599-1168 is shown in FIG. 63 (SEQID NO:174). Clone DNA35599-1168 contains a single open reading framewith an apparent translational initiation site at nucleotide positions69-71 and ending at the stop codon at nucleotide positions 1977-1979(FIG. 63). The predicted polypeptide precursor is 636 amino acids long(FIG. 64). Clone DNA35599-1168 has been deposited with ATCC and isassigned ATCC deposit no. ATCC 209373.

The entire nucleotide sequence of DNA36992-1168 is shown in FIG. 65 (SEQID NO:176). Clone DNA36992-1168 contains a single open reading framewith an apparent translational initiation site at nucleotide positions240-242 and ending at the stop codon at nucleotide positions 2202-2204(FIG. 65). The predicted polypeptide precursor is 654 amino acids long(FIG. 66). Clone DNA36992-1168 has been deposited with ATCC and isassigned ATCC deposit no. ATCC 209382.

Analysis of the amino acid sequence of the full-length PRO236 and PRO262polypeptides suggests that portions of those polypeptides possesssignificant homology to β-galactosidase proteins derived from varioussources, thereby indicating that PRO236 and PRO262 may be novelβ-galactosidase homologs.

Example 30 Isolation of cDNA Clones Encoding Human PRO239

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA30909. Based on the DNA30909 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO239.

A pair of PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-CCTCCCTCTATTACCCATGTC-3′ (SEQ ID NO:186) reversePCR primer 5′-GACCAACTTTCTCTGGGAGTGAGG-3′ (SEQ ID NO:187)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA30909 sequence which had the followingnucleotide sequencehybridization probe5′-GTCACTTTATTTCTCTAACAACAAGCTCGAATCCTTACCAGTGGCAG-3′ (SEQ ID NO:188)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO239 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetallung tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO239 [herein designated as DNA34407-1169](SEQ ID NO:184) and the derived protein sequence for PRO239.

The entire nucleotide sequence of DNA34407-1169 is shown in FIG. 67 (SEQID NO:184). Clone DNA34407-1169 contains a single open reading framewith an apparent translational initiation site at nucleotide positions72-74 and ending at the stop codon at nucleotide positions 1575-1577(FIG. 67). The predicted polypeptide precursor is 501 amino acids long(FIG. 68). Clone DNA34407-1169 has been deposited with ATCC and isassigned ATCC deposit no.ATCC 209383.

Analysis of the amino acid sequence of the full-length PRO239polypeptide suggests that portions of it possess significant homology tothe densin protein, thereby indicating that PRO239 may be a novelmolecule in the densin family.

Example 31 Isolation of cDNA Clones Encoding Human PRO257

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA28731. Based on the DNA28731 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO257.

A pair of PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-TCTCTATTCCAAACTGTGGCG-3′ (SEQ ID NO:191) reversePCR primer 5′-TTTGATGACGATTCGAAGGTGG-3′ (SEQ ID NO:192)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA28731 sequence which had the followingnucleotide sequencehybridization probe5′-GGAAGGATCCTTCACCAGCCCCAATTACCCAAAGCCGCATCCTGAGC-3′   (SEQ ID NO:193)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO257 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalkidney tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO257 [herein designated as DNA35841-1173(SEQ ID NO:189) and the derived protein sequence for PRO257.

The entire nucleotide sequence of DNA35841-1173 is shown in FIG. 69 (SEQID NO:189). Clone DNA35841-1173 contains a single open reading framewith an apparent translational initiation site at nucleotide positions964-966 and ending at the stop codon at nucleotide positions 2785-2787(FIG. 69). The predicted polypeptide precursor is 607 amino acids long(FIG. 70). Clone DNA35841-1173 has been deposited with ATCC and isassigned ATCC deposit no. ATCC 209403.

Analysis of the amino acid sequence of the full-length PRO257polypeptide suggests that portions of it possess significant homology tothe ebnerin protein, thereby indicating that PRO257 may be a novelprotein member related to the ebnerin protein.

Example 32 Isolation of cDNA Clones Encoding Human PRO260

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA30834. Based on the DNA30834 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO260.

PCR primers (forward and two reverse) were synthesized:

forward PCR primer: 5′-TGGTTTGACCAGGCCAAGTTCGG-3′ (SEQ ID NO:196);reverse PCR primer A: 5′-GGATTCATCCTCAAGGAAGAGCGG-3′ (SEQ ID NO:197);and reverse PCR primer B: 5′-AACTTGCAGCATCAGCCACTCTGC-3′ (SEQ ID NO:198)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA30834 sequence which had the followingnucleotide sequence:hybridization probe:5′-TTCCGTGCCCAGCTTCGGTAGCGAGTGGTTCTGGTGGTATTGGCA-3′   (SEQ ID NO:199)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO260 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalkidney tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO260 [herein designated as DNA33470-1175](SEQ ID NO:194) and the derived protein sequence for PRO260.

The entire nucleotide sequence of DNA33470-1175 is shown in FIG. 71 (SEQID NO:194). Clone DNA33470-1175 contains a single open reading framewith an apparent translational initiation site at nucleotide positions67-69 and ending at the stop codon 1468-1470 (see FIG. 71). Thepredicted polypeptide precursor is 467 amino acids long (FIG. 72). CloneDNA33470-1175 has been deposited with ATCC and is assigned ATCC depositno. ATCC 209398.

Analysis of the amino acid sequence of the full-length PRO260polypeptide suggests that portions of it possess significant homology tothe alpha-1-fucosidase precursor, thereby indicating that PRO260 may bea novel fucosidase.

Example 33 Isolation of cDNA Clones Encoding Human PRO263

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA30914. Based on the DNA30914 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO263.

PCR primers (tow forward and one reverse) were synthesized:

forward PCR primer 1: 5′-GAGCTTTCCATCCAGGTGTCATGC-3′ (SEQ ID NO:202);forward PCR primer 2: 5′-GTCAGTGACAGTACCTACTCGG-3′ (SEQ ID NO:203);reverse PCR primer: 5′-TGGAGCAGGAGGAGTAGTAGTAGG-3′ (SEQ ID NO:204)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA30914 sequence which had the followingnucleotide sequence:hybridization probe:5′-AGGAGGCCTGTAGGCTGCTGGGACTAAGTTTGGCCGGCAAGGACCAAGTT-3′   (SEQ IDNO:205)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO263 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalliver tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO263 [herein designated as DNA34431-1177](SEQ ID NO:200) and the derived protein sequence for PRO263.

The entire nucleotide sequence of DNA34431-1177 is shown in FIG. 73 (SEQID NO:200). Clone DNA34431-1177 contains a single open reading framewith an apparent translational initiation site at nucleotide positions160-162 of SEQ ID NO:200 and ending at the stop codon after thenucleotide at position 1126-1128 of SEQ ID NO:200 (FIG. 73). Thepredicted polypeptide precursor is 322 amino acids long (FIG. 74). CloneDNA34431-1177 has been deposited with ATCC and is assigned ATCC depositno. ATCC 209399.

Analysis of the amino acid sequence of the full-length PRO263polypeptide suggests that portions of it possess significant homology toCD44 antigen, thereby indicating that PRO263 may be a novel cell surfaceadhesion molecule.

Example 34 Isolation of cDNA Clones Encoding Human PRO270

A consensus DNA sequence was assembled relative to the other identifiedEST sequences as described in Example 1 above, wherein the consensussequence was designated herein as DNA35712. Based on the DNA35712consensus sequence, oligonucleotides were synthesized: 1) to identify byPCR a cDNA library that contained the sequence of interest, and 2) foruse as probes to isolate a clone of the full-length coding sequence forPRO270. Forward and reverse PCR primers were synthesized:

forward PCR primer (.f1) 5′-GCTTGGATATTCGCATGGGCCTAC-3′ (SEQ ID NO:208)forward PCR primer (.f2) 5′-TGGAGACAATATCCCTGAGG-3′ (SEQ ID NO:209)reverse PCR primer (.r1) 5′-AACAGTTGGCCACAGCATGGCAGG-3′ (SEQ ID NO:210)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA35712 sequence which had the followingnucleotide sequencehybridization probe5′-CCATTGATGAGGAACTAGAACGGGACAAGAGGGTCACTTGGATTGTGGAG-3′ (SEQ ID NO:211)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO270 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetallung tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO270 [herein designated as DNA39510-1181](SEQ ID NO:206) and the derived protein sequence for PRO270.

The entire nucleotide sequence of DNA39510-1181 is shown in FIG. 75 (SEQID NO:206). Clone DNA39510-1181 contains a single open reading framewith an apparent translational initiation site at nucleotide positions3-5 and ending at the stop codon at nucleotide positions 891-893 (FIG.75; SEQ ID NO:206). The predicted polypeptide precursor is 296 aminoacids long (FIG. 76). Clone DNA39510-1181 has been deposited with ATCCand is assigned ATCC deposit no. ATCC 209392.

Analysis of the amino acid sequence of the full-length PRO270 suggeststhat portions of it possess significant homology to thethioredoxin-protein, thereby indicating that the PRO270 protein may be anovel member of the thioredoxin family.

Example 35 Isolation of cDNA Clones Encoding Human PRO271

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA35737. Based on the DNA35737 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO271.

Forward and reverse PCR primers were synthesized:

forward PCR primer 1 5′-TGCTTCGCTACTGCCCTC-3′ (SEQ ID NO:214) forwardPCR primer 2 5′-TTCCCTTGTGGGTTGGAG-3′ (SEQ ID NO:215) forward PCR primer3 5′-AGGGCTGGAAGCCAGTTC-3′ (SEQ ID NO:216) reverse PCR primer 15′-AGCCAGTGAGGAAATGCG-3′ (SEQ ID NO:217) reverse PCR primer 25′-TGTCCAAAGTACACACACCTGAGG-3′ (SEQ ID NO:218)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA35737 sequence which had the followingnucleotide sequencehybridization probe5′-GATGCCACGATCGCCAAGGTGGGACAGCTCTTTGCCGCCTGGAAG-3′   (SEQ ID NO:219)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO271 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalbrain tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO271 [herein designated as DNA39423-1182](SEQ ID NO:212) and the derived protein sequence for PRO271.

The entire nucleotide sequence of DNA39423-1182 is shown in FIG. 77 (SEQID NO:212). Clone DNA39423-1182 contains a single open reading framewith an apparent translational initiation site at nucleotide positions101-103 and ending at the stop codon at nucleotide positions 1181-1183(FIG. 77). The predicted polypeptide precursor is 360 amino acids long(FIG. 78). Clone DNA39423-1182 has been deposited with ATCC and isassigned ATCC deposit no. ATCC 209387.

Analysis of the amino acid sequence of the full-length PRO271polypeptide suggests that it possess significant homology to theproteoglycan link protein, thereby indicating that PRO271 may be a linkprotein homolog.

Example 36 Isolation of cDNA Clones Encoding Human PRO272

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA36460. Based on the DNA36460 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO272.

Forward and reverse PCR primers were synthesized:

forward PCR primer (.f1) 5′-CGCAGGCCCTCATGGCCAGG-3′ (SEQ ID NO:222)forward PCR primer (.f2) 5′-GAAATCCTGGGTAATTGG-3′ (SEQ ID NO:223)reverse PCR primer 5′-GTGCGCGGTGCTCACAGCTCATC-3′ (SEQ ID NO:224)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA36460 sequence which had the followingnucleotide sequencehybridization probe5′-CCCCCCTGAGCGACGCTCCCCCATGATGACGCCCACGGGAACTTC-3′   (SEQ ID NO:225)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pairs identified above. A positive library was then used toisolate clones encoding the PRO272 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetallung tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO272 [herein designated as DNA40620-1183](SEQ ID NO:220) and the derived protein sequence for PRO272.

The entire nucleotide sequence of DNA40620-1183 is shown in FIG. 79 (SEQID NO:220). Clone DNA40620-1183 contains a single open reading framewith an apparent translational initiation site at nucleotide positions35-37 and ending at the stop codon at nucleotide positions 1019-1021(FIG. 79). The predicted polypeptide precursor is 328 amino acids long(FIG. 80). Clone DNA40620-1183 has been deposited with ATCC and isassigned ATCC deposit no. ATCC 209388.

Analysis of the amino acid sequence of the full-length PRO272polypeptide suggests that portions of it possess significant homology tothe human and mouse reticulocalbin proteins, respectively, therebyindicating that PRO272 may be a novel reticulocalbin protein.

Example 37 Isolation of cDNA Clones Encoding Human PRO294

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA35731. Based on the DNA35731 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO294.

Forward and reverse PCR primers were synthesized:

forward PCR primer (.f1) 5′-TGGTCTCGCACACCGATC-3′ (SEQ ID NO:228)forward PCR primer (.f2) 5′-CTGCTGTCCACAGGGGAG-3′ (SEQ ID NO:229)forward PCR primer (.f3) 5′-CCTTGAAGCATACTGCTC-3′ (SEQ ID NO:230)forward PCR primer (.f4) 5′-GAGATAGCAATTTCCGCC-3′ (SEQ ID NO:231)reverse PCR primer (.r1) 5′-TTCCTCAAGAGGGCAGCC-3′ (SEQ ID NO:232)reverse PCR primer (.r2) 5′-CTTGGCACCAATGTCCGAGATTTC-3′ (SEQ ID NO:233)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA35731 sequence which had the followingnucleotide sequencehybridization probe5′-GCTCTGAGGAAGGTGACGCGCGGGGCCTCCGAACCCTTGGCCTTG-3′ (SEQ ID NO:234)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pairs identified above. A positive library was then used toisolate clones encoding the PRO294 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalbrain tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO294 [herein designated as DNA40604-1187](SEQ ID NO:226) and the derived protein sequence for PRO294.

The entire nucleotide sequence of DNA40604-1187 is shown in FIG. 81 (SEQID NO:226). Clone DNA40604-1187 contains a single open reading framewith an apparent translational initiation site at nucleotide positions396-398 and ending at the stop codon at nucleotide positions 2046-2048(FIG. 81). The predicted polypeptide precursor is 550 amino acids long(FIG. 82). Clone DNA40604-1187 has been deposited with ATCC and isassigned ATCC deposit no. 209394.

Analysis of the amino acid sequence of the full-length PRO294polypeptide suggests that portions of it possess significant homology toportions of various collagen proteins, thereby indicating that PRO294may be collagen-like molecule.

Example 38 Isolation of cDNA Clones Encoding Human PRO295

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA35814. Based on the DNA35814 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO295.

Forward and reverse PCR primers were synthesized:

forward PCR primer (.f1) 5′-GCAGAGCGGAGATGCAGCGGTTG-3′ (SEQ ID NO:238)forward PCR primer (.f2) 5′-CCCAGCATGTACTGCCAG-3′ (SEQ ID NO:239)forward PCR primer (.f3) 5′-TTGGCAGCTTCATGGAGG-3′ (SEQ ID NO:240)forward PCR primer (.f4) 5′-CCTGGGCAAAAATGCAAC-3′ (SEQ ID NO:241)reverse PCR primer (.r1) 5′-CTCCAGCTCCTGGCGCACCTCCTC-3′ (SEQ ID NO:242)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA35814 sequence which had the followingnucleotide sequencehybridization probe5′-GGCTCTCAGCTACCGCGCAGGAGCGAGGCCACCCTCAATGAGATG-3′ (SEQ ID NO:243)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pairs identified above. A positive library was then used toisolate clones encoding the PRO295 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetallung tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO295 [herein designated as DNA38268-1188](SEQ ID NO:235) and the derived protein sequence for PRO295.

The entire nucleotide sequence of DNA38268-1188 is shown in FIG. 83 (SEQID NO:235). Clone DNA38268-1188 contains a single open reading framewith an apparent translational initiation site at nucleotide positions153-155 and ending at the stop codon at nucleotide positions 1202-1204(FIG. 83). The predicted polypeptide precursor is 350 amino acids long(FIG. 84). Clone DNA38268-1188 has been deposited with ATCC and isassigned ATCC deposit no. 209421.

Analysis of the amino acid sequence of the full-length PRO295polypeptide suggests that portions of it possess significant homology tothe integrin proteins, thereby indicating that PRO295 may be a novelintegrin.

Example 39 Isolation of cDNA Clones Encoding Human PRO293

The extracellular domain (ECD) sequences (including the secretionsignal, if any) of from about 950 known secreted proteins from theSwiss-Prot public protein database were used to search expressedsequence tag (EST) databases. The EST databases included public ESTdatabases (e.g., GenBank) and a proprietary EST DNA database (LIFESEQ™,Incyte Pharmaceuticals, Palo Alto, Calif.). The search was performedusing the computer program BLAST or BLAST2 (Altshul et al., Methods inEnzymology 266:460-480 (1996)) as a comparison of the ECD proteinsequences to a 6 frame translation of the EST sequence. Thosecomparisons resulting in a BLAST score of 70 (or in some cases 90) orgreater that did not encode known proteins were clustered and assembledinto consensus DNA sequences with the program “phrap” (Phil Green,University of Washington, Seattle, Wash.).

Based on an expression tag sequence designated herein as T08294identified in the above analysis, oligonucleotides were synthesized: 1)to identify by PCR a cDNA library that contained the sequence ofinterest, and 2) for use as probes to isolate a clone of the full-lengthcoding sequence for PRO293.

A pair of PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-AACAAGGTAAGATGCCATCCTG-3′ (SEQ ID NO:246) reversePCR primer 5′-AAACTTGTCGATGGAGACCAGCTC-3′ (SEQ ID NO:247)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the expression sequence tag which had the followingnucleotide sequencehybridization probe5′-AGGGGCTGCAAAGCCTGGAGAGCCTCTCCTTCTATGACAACCAGC-3′   (SEQ ID NO:248)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO293 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalbrain tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO293 [herein designated as DNA37151-1193](SEQ ID NO:244) and the derived protein sequence for PRO293.

The entire nucleotide sequence of DNA37151-1193 is shown in FIG. 85 (SEQID NO:244). Clone DNA37151-1193 contains a single open reading framewith an apparent translational initiation site at nucleotide positions881-883 and ending at the stop codon after nucleotide position 3019 ofSEQ ID NO:244, FIG. 85). The predicted polypeptide precursor is 713amino acids long (FIG. 86). Clone DNA37151-1193 has been deposited withATCC and is assigned ATCC deposit no. ATCC 209393.

Analysis of the amino acid sequence of the full-length PRO293polypeptide suggests that portions of it possess significant homology tothe NLRR proteins, thereby indicating that PRO293 may be a novel NLRRprotein.

Example 40 Isolation of cDNA Clones Encoding Human PRO247

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA33480. Based on the DNA33480 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO247.

A pair of PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-CAACAATGAGGGCACCAAGC-3′ (SEQ ID NO:251) reversePCR primer 5′-GATGGCTAGGTTCTGGAGGTTCTG-3′ (SEQ ID NO:252)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the DNA33480 expression sequence tag which had thefollowing nucleotide sequencehybridization probe5′-CAACCTGCAGGAGATTGACCTCAAGGACAACAACCTCAAGACCATCG-3′   (SEQ ID NO:253)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO247 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalbrain tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO247 [herein designated as DNA35673-1201](SEQ ID NO:249) and the derived protein sequence for PRO247.

The entire nucleotide sequence of DNA35673-1201 is shown in FIG. 89 (SEQID NO:249). Clone DNA35673-1201 contains a single open reading framewith an apparent translational initiation site at nucleotide positions80-82 of SEQ ID NO:249 and ending at the stop codon after nucleotideposition 1717 of SEQ ID NO:249 (FIG. 89). The predicted polypeptideprecursor is 546 amino acids long (FIG. 88). Clone DNA35673-1201 hasbeen deposited with ATCC and is assigned ATCC deposit no. 209418.

Analysis of the amino acid sequence of the full-length PRO247polypeptide suggests that portions of it possess significant homology tothe densin molecule and KIAA0231, thereby indicating that PRO247 may bea novel leucine rich repeat protein.

Example 41 Isolation of cDNA Clones Encoding Human PRO302. PRO303.PRO304. PRO307 and PRO343

Consensus DNA sequences were assembled relative to other EST sequencesusing phrap as described in Example 1 above. These consensus sequencesare herein designated DNA35953, DNA35955, DNA35958, DNA37160 andDNA30895. Based on the DNA35953 consensus sequence, oligonucleotideswere synthesized: 1) to identify by PCR a cDNA library that containedthe sequence of interest, and 2) for use as probes to isolate a clone ofthe full-length coding sequence for PRO302.

PCR primers (forward and reverse) were synthesized:

forward PCR primer 1 5′-GTCCGCAAGGATGCCTACATGTTC-3′ (SEQ ID NO:264)forward PCR primer 2 5′-GCAGAGGTGTCTAAGGTTG-3′ (SEQ ID NO:265) reversePCR primer 5′-AGCTCTAGACCAATGCCAGCTTCC-3′ (SEQ ID NO:266)Also, a synthetic oligonucleotide hybridization probe was constructedfrom the consensus DNA35953 sequence which had the following nucleotidesequencehybridization probe5′-GCCACCAACTCCTGCAAGAACTTCTCAGAACTGCCCCTGGTCATG-3′   (SEQ ID NO:267)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pairs identified above. A positive library was then used toisolate clones encoding the PRO302 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalkidney tissue (LIB228).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO302 [herein designated as DNA40370-1217](SEQ ID NO:254) and the derived protein sequence for PRO302.

The entire nucleotide sequence of DNA40370-1217 is shown in FIG. 89 (SEQID NO:254). Clone DNA40370-1217 contains a single open reading framewith an apparent translational initiation site at nucleotide positions34-36 and ending at the stop codon at nucleotide positions 1390-1392(FIG. 89). The predicted polypeptide precursor is 452 amino acids long(FIG. 90). Various unique aspects of the PRO302 protein are shown inFIG. 90. Clone DNA40370-1217 has been deposited with the ATCC on Nov.21, 1997 and is assigned ATCC deposit no. ATCC 209485.

Based on the DNA35955 consensus sequence, oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence for PRO303.

A pair of PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-GGGGAATTCACCCTATGACATTGCC-3′ (SEQ ID NO:268)reverse PCR primer 5′-GAATGCCCTGCAAGCATCAACTGG-3′ (SEQ ID NO:269)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA35955 sequence which had the followingnucleotide sequence:hybridization probe5′-GCACCTGTCACCTACACTAAACACATCCAGCCCATCTGTCTCCAGGCCTC-3′   (SEQ IDNO:270)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pairs identified above. A positive library was then used toisolate clones encoding the PRO303 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetallung tissue (L1125).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO303 [herein designated as DNA42551-1217](SEQ ID NO:256) and the derived protein sequence for PRO303.

The entire nucleotide sequence of DNA42551-1217 is shown in FIG. 91 (SEQID NO:256). Clone DNA42551-1217 contains a single open reading framewith an apparent translational initiation site at nucleotide positions20-22 and ending at the stop codon at nucleotide positions 962-964 (FIG.91). The predicted polypeptide precursor is 314 amino acids long (FIG.92). Various unique aspects of the PRO303 protein are shown in FIG. 92.Clone DNA42551-1217 has been deposited on Nov. 21, 1997 with the ATCCand is assigned ATCC deposit no. ATCC 209483.

Based on the DNA35958 consensus sequence, oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence for PRO304.

Pairs of PCR primers (forward and reverse) were synthesized:

forward PCR primer 1 5′-GCGGAAGGGCAGAATGGGACTCCAAG-3′ (SEQ ID NO:271)forward PCR primer 2 5′-CAGCCCTGCCACATGTGC-3′ (SEQ ID NO:272) forwardPCR primer 3 5′-TACTGGGTGGTCAGCAAC-3′ (SEQ ID NO:273) reverse PCR primer5′-GGCGAAGAGCAGGGTGAGACCCCG-3′ (SEQ ID NO:274)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA35958 sequence which had the followingnucleotide sequencehybridization probe5′-GCCCTCATCCTCTCTGGCAAATGCAGTTACAGCCCGGAGCCCGAC-3′   (SEQ ID NO:275)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pairs identified above. A positive library was then used toisolate clones encoding the PRO304 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from 22 weekhuman fetal brain tissue (LIB153).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO304 [herein designated as DNA39520-1217](SEQ ID NO:258) and the derived protein sequence for PRO304.

The entire nucleotide sequence of DNA39520-1217 is shown in FIG. 93 (SEQID NO:258). Clone DNA39520-1217 contains a single open reading framewith an apparent translational initiation site at nucleotide positions34-36 and ending at the stop codon at nucleotide positions 1702-1704(FIG. 93). The predicted polypeptide precursor is 556 amino acids long(FIG. 94). Various unique aspects of the PRO304 protein are shown inFIG. 94. Clone DNA39520-1217 has been deposited with ATCC on Nov. 21,1997 and is assigned ATCC deposit no. ATCC 209482.

Based on the DNA37160 consensus sequence, oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence for PRO307.

Pairs of PCR primers (forward and reverse) were synthesized:

forward PCR primer 1 5′-GGGCAGGGATTCCAGGGCTCC-3′ (SEQ ID NO:276) forwardPCR primer 2 5′-GGCTATGACAGCAGGTTC-3′ (SEQ ID NO:277) forward PCR primer3 5′-TGACAATGACCGACCAGG-3′ (SEQ ID NO:278) reverse PCR primer5′-GCATCGCATTGCTGGTAGAGCAAG-3′ (SEQ ID NO:279)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA37160 sequence which had the followingnucleotide sequencehybridization probe5′-TTACAGTGCCCCCTGGAAACCCACTTGGCCTGCATACCGCCTCCC-3′   (SEQ ID NO:280)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pairs identified above. A positive library was then used toisolate clones encoding the PRO307 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalliver tissue (LIB229).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO307 [herein designated as DNA41225-1217](SEQ ID NO:260) and the derived protein sequence for PRO307.

The entire nucleotide sequence of DNA41225-1217 is shown in FIG. 95 (SEQID NO:260). Clone DNA41225-1217 contains a single open reading framewith an apparent translational initiation site at nucleotide positions92-94 and ending at the stop codon at nucleotide positions 1241-1243(FIG. 95). The predicted polypeptide precursor is 383 amino acids long(FIG. 96). Various unique aspects of the PRO307 protein are shown inFIG. 96. Clone DNA41225-1217 has been deposited with ATCC on Nov. 21,1997 and is assigned ATCC deposit no. ATCC 209491.

Based on the DNA30895 consensus sequence, oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence for PRO343.

A pair of PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-CGTCTCGAGCGCTCCATACAGTTCCCTTGCCCCA-3′ (SEQ IDNO:281) reverse PCR primer 5′-TGGAGGGGGAGCGGGATGCTTGTCTGGGCGACTCCGGGGGCC(SEQ ID NO:282) CCCTCATGTGCCAGGTGGA-3′Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA30895 sequence which had the followingnucleotide sequence

hybridization probe5′-CCCTCAGACCCTGCAGAAGCTGAAGGTTCCTATCATCGACTCGGAAGTCTGCAGCCATCTG (SEQ IDNO:283) TACTGGCGGGGAGCAGGACAGGGACCCATCACTGAGGACATGCTGTGTGCCGGCTACT-3′

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pairs identified above. A positive library was then used toisolate clones encoding the PRO343 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetallung tissue (LIB26).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO343 [herein designated as DNA43318-1217](SEQ ID NO:262) and the derived protein sequence for PRO343.

The entire nucleotide sequence of DNA43318-1217 is shown in FIG. 97 (SEQID NO:262). Clone DNA43318-1217 contains a single open reading framewith an apparent translational initiation site at nucleotide positions53-55 and ending at the stop codon at nucleotide positions 1004-1006(FIG. 97). The predicted polypeptide precursor is 317 amino acids long(FIG. 98). Various unique aspects of the PRO343 protein are shown inFIG. 98. Clone DNA43318-1217 has been deposited with ATCC on Nov. 21,1997 and is assigned ATCC deposit no. ATCC 209481.

Example 42 Isolation of cDNA Clones Encoding Human PRO328

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA35615. Based on the DNA35615 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO328.

Forward and reverse PCR primers were synthesized:

forward PCR primer 5′-TCCTGCAGTTTCCTGATGC-3′ (SEQ ID NO:286) reverse PCRprimer 5′-CTCATATTGCACACCAGTAATTCG-3′ (SEQ ID NO:287)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA35615 sequence which had the followingnucleotide sequencehybridization probe5′-ATGAGGAGAAACGTTTGATGGTGGAGCTGCACAACCTCTACCGGG-3′ (SEQ ID NO:288)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO328 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalkidney tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO328 [herein designated as DNA40587-1231](SEQ ID NO:284) and the derived protein sequence for PRO328.

The entire nucleotide sequence of DNA40587-1231 is shown in FIG. 99 (SEQID NO:284). Clone DNA40587-1231 contains a single open reading framewith an apparent translational initiation site at nucleotide positions15-17 and ending at the stop codon at nucleotide positions 1404-1406(FIG. 99). The predicted polypeptide precursor is 463 amino acids long(FIG. 100). Clone DNA40587-1231 has been deposited with ATCC and isassigned ATCC deposit no. ATCC 209438.

Analysis of the amino acid sequence of the full-length PRO328polypeptide suggests that portions of it possess significant homology tothe human glioblastoma protein and to the cysteine rich secretoryprotein thereby indicating that PRO328 may be a novel glioblastomaprotein or cysteine rich secretory protein.

Example 43 Isolation of cDNA Clones Encoding Human PRO335, PRO331 orPRO326

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA36685. Based on the DNA36685 consensus sequence,and Incyte EST sequence no. 2228990, oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence for PRO335, PRO331 or PRO326.

Forward and reverse PCR primers were synthesized for the determinationof PRO335:

forward PCR primer 5′-GGAACCGAATCTCAGCTA-3′ (SEQ ID NO:295) forward PCRprimer 5′-CCTAAACTGAACTGGACCA-3′ (SEQ ID NO:296) forward PCR Primer5′-GGCTGGAGACACTGAACCT-3′ (SEQ ID NO:297) forward PCR primer5′-ACAGCTGCACAGCTCAGAACAGTG-3′ (SEQ ID NO:298) reverse PCR primer5′-CATTCCCAGTATAAAAATTTTC-3′ (SEQ ID NO:299) reverse PCR primer5′-GGGTCTTGGTGAATGAGG-3′ (SEQ ID NO:300) reverse PCR primer5′-GTGCCTCTCGGTTACCACCAATGG-3′ (SEQ ID NO:301)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed for the determination of PRO335 which had the followingnucleotide sequencehybridization probe5′-GCGGCCACTGTTGGACCGAACTGTAACCAAGGGAGAAACAGCCGTCCTAC-3′ (SEQ ID NO:302)

Forward and reverse PCR primers were synthesized for the determinationof PRO331:

forward PCR primer 5′-GCCTTTGACAACCTTCAGTCACTAGTGG-3′ (SEQ ID NO:303)reverse PCR primer 5′-CCCCATGTGTCCATGACTGTTCCC-3′ (SEQ ID NO:304)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed for the determination of PRO331 which bad the followingnucleotide sequencehybridization probe5′-TACTGCCTCATGACCTCTTCACTCCCTTGCATCATCTTAGAGCGG-3′ (SEQ ID NO:305)

Forward and reverse PCR primers were synthesized for the determinationof PRO326:

forward PCR primer 5′-ACTCCAAGGAAATCGGATCCGTTC-3′ (SEQ ID NO:306)reverse PCR primer 5′-TTAGCAGCTGAGGATGGGCACAAC-3′ (SEQ ID NO:307)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed for the determination of PRO331 which had the followingnucleotide sequencehybridization probe5′-GCCTTCACTGGTTTGGATGCATTGGAGCATCTAGACCTGAGTGACAACGC-3′ (SEQ ID NO:308)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pairs identified above. A positive library was then used toisolate clones encoding the PRO335, PRO331 or PRO326 gene using theprobe oligonucleotide and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalkidney tissue (PRO335 and PRO326) and human fetal brain (PRO331).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO335, PRO331 or PRO326 [herein designatedas SEQ ID NOS:289, 291 and 293, respectively; see FIGS. 101, 103 and105, respectively], and the derived protein sequence for PRO335, PRO331or PRO326 (see FIGS. 102, 104 and 106, respectively; SEQ ID NOS:290, 292and 294, respectively).

The entire nucleotide sequences are shown in FIGS. 101, 103 and 105,deposited with the ATCC on Jun. 2, 1998, Nov. 7, 1997 and Nov. 21, 1997,respectively.

Analysis of the amino acid sequence of the full-length PRO335, PRO331 orPRO326 polypeptide suggests that portions of it possess significanthomology to the LIG-1 protein, thereby indicating that PRO335, PRO331and PRO326 may be a novel LIG-1-related protein.

Example 44 Isolation of cDNA clones Encoding Human PRO332

Based upon an ECD homology search performed as described in Example 1above, a consensus DNA sequence designated herein as DNA36688 wasassembled. Based on the DNA36688 consensus sequence, oligonucleotideswere synthesized to identify by PCR a cDNA library that contained thesequence of interest and for use as probes to isolate a clone of thefull-length coding sequence for PRO332.

A pair of PCR primers (forward and reverse) were synthesized:

5′-GCATTGGCCGCGAGACTTTGCC-3′ (SEQ ID NO:311)5′-GCGGCCACGGTCCTTGGAAATG-3′ (SEQ ID NO:312)

A probe was also synthesized:5′-TGGAGGAGCTCAACCTCAGCTACAACCGCATCACCAGCCCACAGG-3′ (SEQ ID NO:313)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO332 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from a humanfetal liver library (LIB229).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for DNA40982-1235 and the derived proteinsequence for PRO332.

The entire nucleotide sequence of DNA40982-1235 is shown in FIG. 107(SEQ ID NO:309). Clone DNA40982-1235 contains a single open readingframe (with an apparent translational initiation site at nucleotidepositions 342-344, as indicated in FIG. 107). The predicted polypeptideprecursor is 642 amino acids long, and has a calculated molecular weightof 72,067 (p1: 6.60). Clone DNA40982-1235 has been deposited with ATCCand is assigned ATCC deposit no. ATCC 209433.

Based on a BLAST and FastA sequence alignment analysis of thefull-length sequence, PRO332 shows about 30-40% amino acid sequenceidentity with a series of known proteoglycan sequences, including, forexample, fibromodulin and fibromodulin precursor sequences of variousspecies (FMOD_BOVIN, FMOD CHICK, FMOD_RAT, FMOD_MOUSE, FMOD_HUMAN,P_R36773), osteomodulin sequences (AB0001141 1, AB007848_(—)1), decorinsequences (CFU83141_(—)1, OCU03394_(—)1, P_R42266, P_R42267, P_R42260,P^(—)R89439), keratan sulfate proteoglycans (BTU48360_(—)1,AF022890_(—)1), corneal proteoglycan (AF022256_(—)1), and bone/cartilageproteoglycans and proteoglycane precursors (PGS1_BOVIN, PGS2_MOUSE,PGS2_HUMAN).

Example 45 Isolation of cDNA clones Encoding Human PRO334

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. Based on the consensussequence, oligonucleotides were synthesized: 1) to identify by PCR acDNA library that contained the sequence of interest, and 2) for use asprobes to isolate a clone of the full-length coding sequence for PRO334.

Forward and reverse PCR primers were synthesized for the determinationof PRO334:

forward PCR primer 5′-GATGGTTCCTGCTCAAGTGCCCTG-3′ (SEQ ID NO:316)reverse PCR primer 5′-TTGCACTTGTAGGACCCACGTACG-3′ (SEQ ID NO:317)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed for the determination of PRO334 which had the followingnucleotide sequencehybridization probe5′-CTGATGGGAGGACCTGTGTAGATGTTGATGAATGTGCTACAGGAAGAGCC-3′ (SEQ ID NO:318)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO334 gene using the probe oligonucleotideand one of the PCR primers.

Human fetal kidney cDNA libraries used to isolate the cDNA clones wereconstructed by standard methods using commercially available reagentssuch as those from Invitrogen, San Diego, Calif.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO334 [herein designated as DNA41379-1236](SEQ ID NO:314) and the derived protein sequence for PRO334.

The entire nucleotide sequence of DNA41379-1236 (also referred to asUNQ295) is shown in FIG. 109 (SEQ ID NO:314). Clone DNA41379-1236contains a single open reading frame with an apparent translationalinitiation site at nucleotide positions 203-205 and ending at the stopcodon at nucleotide positions 1730-1732 (FIG. 109). The predictedpolypeptide precursor is 509 amino acids long (FIG. 110). CloneDNA41379-1236 has been deposited with ATCC and is assigned ATCC depositno. ATCC 209488.

Analysis of the amino acid sequence of the full-length PRO334polypeptide suggests that portions of it possess significant homology tothe fibulin and fibrillin proteins, thereby indicating that PRO334 maybe a novel member of the EGF protein family.

Example 46 Isolation of cDNA Clones Encoding Human PRO346

A consensus DNA sequence was identified using phrap as described inExample 1 above. Specifically, this consensus sequence is hereindesignated DNA38240. Based on the DNA38240 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length PRO346 coding sequence.

RNA for construction of the cDNA libraries was isolated from human fetalliver. The cDNA libraries used to isolated the cDNA clones wereconstructed by standard methods using commercially available reagents(e.g., Invitrogen, San Diego, Calif.; Clontech, etc.) The cDNA wasprimed with oligo dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

A cDNA clone was sequenced in entirety. The entire nucleotide sequenceof DNA44167-1243 is shown in FIG. 111 (SEQ ID NO:319). CloneDNA44167-1243 contains a single open reading frame with an apparenttranslational initiation site at nucleotide positions 64-66 (FIG. 111;SEQ ID NO:319). The predicted polypeptide precursor is 450 amino acidslong. Clone DNA44167-1243 has been deposited with ATCC and is assignedATCC deposit no. ATCC 209434 (designation DNA44167-1243).

Based on a BLAST, BLAST-2 and FastA sequence alignment analysis (usingthe ALIGN computer program) of the full-length sequence, PRO346 showsamino acid sequence identity to carcinoembryonic antigen (28%).

The oligonucleotide sequences used in the above procedure were thefollowing:

OLI2691 (38240. f1)

5′-GATCCTGTCACAAAGCCAGTGGTGC-3′ (SEQ ID NO:321) OLI2693 (38240.r1)5′-CACTGACAGGGTTCCTCACCCAGG-3′ (SEQ ID NO:322) OLI2692 (38240.p1)5′-CTCCCTCTGGGCTGTGGAGTATGTGGGGAACATGACCCTGACATG-3′ (SEQ ID NO:323)

Example 47 Isolation of cDNA Clones Encoding Human PRO268

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA35698. Based on the DNA35698 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO268.

Forward and reverse PCR primers were synthesized:

forward PCR primer 1 5′-TGAGGTGGGCAAGCGGCGAAATG-3′ (SEQ ID NO:326)forward PCR primer 2 5′-TATGTGGATCAGGACGTGCC-3′ (SEQ ID NO:327) forwardPCR primer 3 5′-TGCAGGGTTCAGTCTAGATTG-3′ (SEQ ID NO:328) reverse PCRprimer 5′-TTGAAGGACAAAGGCAATCTGCCAC-3′ (SEQ ID NO:329)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA35698 sequence which had the followingnucleotide sequencehybridization probe5′-GGAGTCTTGCAGTTCCCCTGGCAGTCCTGGTGCTGTTGCTTTGGG-3′   (SEQ ID NO:330)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO268 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetallung tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO268 [herein designated as DNA39427-1179](SEQ ID NO:324) and the derived protein sequence for PRO268.

The entire nucleotide sequence of DNA39427-1179 is shown in FIG. 113(SEQ ID NO:324). Clone DNA39427-1179 contains a single open readingframe with an apparent translational initiation site at nucleotidepositions 13-15 and ending at the stop codon at nucleotide positions853-855 (FIG. 113). The predicted polypeptide precursor is 280 aminoacids long (FIG. 114). Clone DNA39427-1179 has been deposited with ATCCand is assigned ATCC deposit no. ATCC 209395.

Analysis of the amino acid sequence of the full-length PRO268polypeptide suggests that it possess significant homology to proteindisulfide isomerase, thereby indicating that PRO268 may be a novelprotein disulfide isomerase.

Example 48 Isolation of cDNA Clones Encoding Human PRO330

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA35730. Based on the DNA35730 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO330.

Forward and reverse PCR primers were synthesized:

forward PCR primer 1 5′-GCAGGCACAATTTCCAGA-3′ (SEQ ID NO:333) forwardPCR primer 2 5′-GGACCCTTCTGTGTGCCAG-3′ (SEQ ID NO:334) reverse PCRprimer 1 5′-GGTCTCAAGAACTCCTGTC-3′ (SEQ ID NO:335) reverse PCR primer 25′-ACACTCAGCATTGCCTGGTACTTG-3′ (SEQ ID NO:336)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus sequence which had the followingnucleotide sequencehybridization probe5′-GGGCACATGACTGACCTGATTTATGCAGAGAAAGAGCTGGTGCAG-3′   (SEQ ID NO:337)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO330 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalliver tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO330 [herein designated as DNA40603-1232](SEQ ID NO:331) and the derived protein sequence for PRO330.

The entire nucleotide sequence of DNA40603-1232 is shown in FIG. 115(SEQ ID NO:331). Clone DNA40603-1232 contains a single open readingframe with an apparent translational initiation site at nucleotidepositions 167-169 and ending at the stop codon at nucleotide positions1766-1768 (FIG. 115). The predicted polypeptide precursor is 533 aminoacids long (FIG. 116). Clone DNA40603-1232 has been deposited with ATCCand is assigned ATCC deposit no.ATCC 209486 on Nov. 21, 1997.

Analysis of the amino acid sequence of the full-length PRO330polypeptide suggests that portions of it possess significant homology tothe mouse prolyl 4-hydroxylase alpha subunit protein, thereby indicatingthat PRO330 may be a novel prolyl 4-hydroxylase alpha subunitpolypeptide.

Example 49 Isolation of cDNA Clones Encoding Human PRO310

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA40553. Based on the DNA40553 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO310.

Forward and reverse PCR primers were synthesized:

forward PCR primer 1 5′-TCCCCAAGCCGTTCTAGACGCGG-3′ (SEQ ID NO:342)forward PCR primer 2 5′-CTGGTTCTTCCTTGCACG-3′ (SEQ ID NO:343) reversePCR primer 5′-GCCCAAATGCCCTAAGGCGGTATACCCC-3′ (SEQ ID NO:344)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus sequence which had the followingnucleotide sequencehybridization probe5′-GGGTGTGATGCTTGGAAGCATTTTCTGTGCTTTGATCACTATGCTAGGAC-3′   (SEQ IDNO:345)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO310 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalliver tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO310 [herein designated as DNA43046-1225(SEQ ID NO:340) and the derived protein sequence for PRO310 (SEQ IDNO:341).

The entire nucleotide sequence of DNA43046-1225 is shown in FIG. 119(SEQ ID NO:340). Clone DNA43046-1225 contains a single open readingframe with an apparent translational initiation site at nucleotidepositions 81-83 and ending at the stop codon at nucleotide positions1035-1037 (FIG. 119). The predicted polypeptide precursor is 318 aminoacids long (FIG. 120) and has a calculated molecular weight ofapproximately 36,382 daltons. Clone DNA43046-1225 has been depositedwith ATCC and is assigned ATCC deposit no. ATCC 209484.

Analysis of the amino acid sequence of the full-length PRO310polypeptide suggests that portions of it possess homology to C. elegansproteins and to fringe, thereby indicating that PRO310 may be involvedin development.

Example 50 Isolation of cDNA clones Encoding Human PRO339

An expressed sequence tag (EST) DNA database (LIFESEQ™, IncytePharmaceuticals, Palo Alto, Calif.) was searched and ESTs wereidentified. An assembly of Incyte clones and a consensus sequence wasformed using phrap as described in Example 1 above.

Forward and reverse PCR primers were synthesized based upon theassembly-created consensus sequence:

forward PCR primer 1 5′-GGGATGCAGGTGGTGTCTCATGGGG-3′ (SEQ ID NO:346)forward PCR primer 2 5′-CCCTCATGTACCGGCTCC-3′ (SEQ ID NO:347) forwardPCR primer 3 5′-GTGTGACACAGCGTGGGC-3′ (SEQ ID NO:43) forward PCR primer4 5′-GACCGGCAGGCTTCTGCG-3′ (SEQ ID NO:44) reverse PCR primer 15′-CAGCAGCTTCAGCCACCAGGAGTGG-3′ (SEQ ID NO:45) reverse PCR primer 25′-CTGAGCCGTGGGCTGCAGTCTCGC-3′ (SEQ ID NO:46)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus sequence which had the followingnucleotide sequencehybridization probe5′-CCGACTACGACTGGTTCTTCATCATGCAGGATGACACATATGTGC-3′   (SEQ ID NO:47)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pairs identified above. A positive library was then used toisolate clones encoding the PRO339 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalliver tissue.

A cDNA clone was sequenced in entirety. The entire nucleotide sequenceof DNA43466-1225 is shown in FIG. 117 (SEQ ID NO:338). CloneDNA43466-1225 contains a single open reading frame with an apparenttranslational initiation site at nucleotide positions 333-335 and endingat the stop codon found at nucleotide positions 2649-2651 (FIG. 117; SEQID NO:338). The predicted polypeptide precursor is 772 amino acids longand has a calculated molecular weight of approximately 86,226 daltons.Clone DNA43466-1225 has been deposited with ATCC and is assigned ATCCdeposit no. ATCC 209490.

Based on a BLAST and FastA sequence alignment analysis (using the ALIGNcomputer program) of the full-length sequence, PRO339 has homology to C.elegans proteins and collagen-like polymer sequences as well as tofringe, thereby indicating that PRO339 may be involved in development ortissue growth.

Example 51 Isolation of cDNA Clones Encoding Human PRO244

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. Based on this consensussequence, oligonucleotides were synthesized to identify by PCR a cDNAlibrary that contained the sequence of interest and for use as probes toisolate a clone of the full-length coding sequence for PRO244.

A pair of PCR primers (forward and reverse) were synthesized:

5′-TTCAGCTTCTGGGATGTAGGG-3′ (30923.f1) (SEQ ID NO:378)5′-TATTCCTACCATTTCACAAATCCG-3′ (30923.r1) (SEQ ID NO:379) A probe wasalso synthesized:5′-GGAGGACTGTGCCACCATGAGAGACTCTTCAAACCCAAGGCAAAATTGG-3′ (30923.p1) (SEQID NO:380)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO244 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from a humanfetal kidney library. DNA sequencing of the clones isolated as describedabove gave the full-length DNA sequence and the derived protein sequencefor PRO244.

The entire nucleotide sequence of PRO244 is shown in FIG. 121 (SEQ IDNO:376). Clone DNA35668-1171 contains a single open reading frame withan apparent translational initiation site at nucleotide positions106-108 (FIG. 121). The predicted polypeptide precursor is 219 aminoacids long. Clone DNA35668-1171 has been deposited with ATCC (designatedas DNA35663-1171) and is assigned ATCC deposit no. ATCC209371. Theprotein has a cytoplasmic domain (aa 1-20), a transmembrane domain (aa21-46), and an extracellular domain (aa 47-219), with a C-lectin domainat aa 55-206.

Based on a BLAST and FastA sequence alignment analysis of thefull-length sequence, PRO244 shows notable amino acid sequence identityto hepatic lectin gallus gallus (43%), HIC hp120-binding C-type lectin(42%), macrophage lectin 2 (HUMHML2-1, 41%), and sequence PR32188 (44%).

Example 52 Use of PRO Polypeptide-Encoding Nucleic Acid as HybridizationProbes

The following method describes use of a nucleotide sequence encoding aPRO polypeptide as a hybridization probe.

DNA comprising the coding sequence of of a PRO polypeptide of interestas disclosed herein may be employed as a probe or used as a basis fromwhich to prepare probes to screen for homologous DNAs (such as thoseencoding naturally-occurring variants of the PRO polypeptide) in humantissue cDNA libraries or human tissue genomic libraries.

Hybridization and washing of filters containing either library DNAs isperformed under the following high stringency conditions. Hybridizationof radiolabeled PRO polypeptide-encoding nucleic acid-derived probe tothe filters is performed in a solution of 50% formamide, 5×SSC, 0.1%SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8,2×Denhardt's solution, and 10% dextran sulfate at 42° C. for 20 hours.Washing of the filters is performed in an aqueous solution of 0.1×SSCand 0.1% SDS at 42° C.

DNAs having a desired sequence identity with the DNA encodingfull-length native sequence PRO polypeptide can then be identified usingstandard techniques known in the art.

Example 53 Expression of PRO Polypeptides in E. coli

This example illustrates preparation of an unglycosylated form of adesired PRO polypeptide by recombinant expression in E. coli.

The DNA sequence encoding the desired PRO polypeptide is initiallyamplified using selected PCR primers. The primers should containrestriction enzyme sites which correspond to the restriction enzymesites on the selected expression vector. A variety of expression vectorsmay be employed. An example of a suitable vector is pBR322 (derived fromE. coli; see Bolivar et al., Gene, 2:95 (1977)) which contains genes forampicillin and tetracycline resistance. The vector is digested withrestriction enzyme and dephosphorylated. The PCR amplified sequences arethen ligated into the vector. The vector will preferably includesequences which encode for an antibiotic resistance gene, a trppromoter, a polyhis leader (including the first six STII codons, polyhissequence, and enterokinase cleavage site), the specific PRO polypeptidecoding region, lambda transcriptional terminator, and an argU gene.

The ligation mixture is then used to transform a selected E. coli strainusing the methods described in Sambrook et al., supra. Transformants areidentified by their ability to grow on LB plates and antibioticresistant colonies are then selected. Plasmid DNA can be isolated andconfirmed by restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid culture medium such asLB broth supplemented with antibiotics. The overnight culture maysubsequently be used to inoculate a larger scale culture. The cells arethen grown to a desired optical density, during which the expressionpromoter is turned on.

After culturing the cells for several more hours, the cells can beharvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized PRO polypeptide can then be purified using a metalchelating column under conditions that allow tight binding of theprotein.

PRO187, PRO317, PRO301, PRO224 and PRO238 were successfully expressed inE. coli in apoly-His tagged form, using the following procedure. The DNAencoding PRO187, PRO317, PRO301, PRO224 or PRO238 was initiallyamplified using selected PCR primers. The primers contained restrictionenzyme sites which correspond to the restriction enzyme sites on theselected expression vector, and other useful sequences providing forefficient and reliable translation initiation, rapid purification on ametal chelation column, and proteolytic removal with enterokinase. ThePCR-amplified, poly-His tagged sequences were then ligated into anexpression vector, which was used to transform an E. coli host based onstrain 52 (W3110 fuhA(tonA) Ion galE rpoHts(htpRts) clpP(lacIq).Transformants were first grown in LB containing 50 mg/ml carbenicillinat 30° C. with shaking until an O.D.600 of 3-5 was reached. Cultureswere then diluted 50-100 fold into CRAP media (prepared by mixing 3.57 g(NH₄)₂SO_(4, 0.71) g sodium citrate.2H2O, 1.07 g KCl, 5.36 g Difco yeastextract, 5.36 g Sheffield hycase SF in 500 mL water, as well as 110 mMMPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgSO₄) and grown forapproximately 20-30 hours at 30° C. with shaking. Samples were removedto verify expression by SDS-PAGE analysis, and the bulk culture iscentrifuged to pellet the cells. Cell pellets were frozen untilpurification and refolding.

E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) wasresuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8buffer. Solid sodium sulfite and sodium tetrathionate is added to makefinal concentrations of 0.1M and 0.02 M, respectively, and the solutionwas stirred overnight at 4° C. This step results in a denatured proteinwith all cysteine residues blocked by sulfitolization. The solution wascentrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. Thesupernatant was diluted with 3-5 volumes of metal chelate column buffer(6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micronfilters to clarify. Depending the clarified extract was loaded onto a 5ml Qiagen Ni-NTA metal chelate column equilibrated in the metal chelatecolumn buffer. The column was washed with additional buffer containing50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. The protein waseluted with buffer containing 250 mM imidazole. Fractions containing thedesired protein were pooled and stored at 4° C. Protein concentrationwas estimated by its absorbance at 280 nm using the calculatedextinction coefficient based on its amino acid sequence.

The proteins were refolded by diluting sample slowly into freshlyprepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCl,2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. Refoldingvolumes were chosen so that the final protein concentration was between50 to 100 micrograms/ml. The refolding solution was stirred gently at 4°C. for 12-36 hours. The refolding reaction was quenched by the additionof TFA to a final concentration of 0.4% (pH of approximately 3). Beforefurther purification of the protein, the solution was filtered through a0.22 micron filter and acetonitrile was added to 2-10% finalconcentration. The refolded protein was chromatographed on a Poros R1/Hreversed phase column using a mobile buffer of 0.1% TFA with elutionwith a gradient of acetonitrile from 10 to 80%. Aliquots of fractionswith A280 absorbance were analyzed on SDS polyacrylamide gels andfractions containing homogeneous refolded protein were pooled.Generally, the properly refolded species of most proteins are eluted atthe lowest conceni rations of acetonitrile since those species are themost compact with their hydrophobic interiors shielded from interactionwith the reversed phase resin. Aggregated species are usually eluted athigher acetonitrile concentrations. In addition to resolving misfoldedforms of proteins from the desired form, the reversed phase step alsoremoves endotoxin from the samples.

Fractions containing the desired folded PRO187, PRO317, PRO301, PRO224and PRO238 proteins, respectively, were pooled and the acetonitrileremoved using a gentle stream of nitrogen directed at the solution.Proteins were formulated into 20 mM Hepes, pH 6.8 with 0.14 M sodiumchloride and 4% mannitol by dialysis or by gel filtration using G25Superfine (Pharmacia) resins equilibrated in the formulation buffer andsterile filtered.

Example 54 Expression of PRO Polypeptides in Mammalian Cells

This example illustrates preparation of a glycosylated form of a desiredPRO polypeptide by recombinant expression in mammalian cells.

The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employedas the expression vector. Optionally, the PRO polypeptide-encoding DNAis ligated into pRK5 with selected restriction enzymes to allowinsertion of the PRO polypeptide DNA using ligation methods such asdescribed in Sambrook et al., supra. The resulting vector is calledpRK5-PRO polypeptide.

In one embodiment, the selected host cells may be 293 cells. Human 293cells (ATCC CCL 1573) are grown to confluence in tissue culture platesin medium such as DMEM supplemented with fetal calf serum andoptionally, nutrient components and/or antibiotics. About 10 μg pRK5-PROpolypeptide DNA is mixed with about 1 μg DNA encoding the VA RNA gene[Thimmappaya et al., Cell, 31:543 (1982)] and dissolved in 500 μl of 1mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl₂. To this mixture is added,dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NAPO₄,and a precipitate is allowed to form for 10 minutes at 25° C. Theprecipitate is suspended and added to the 293 cells and allowed tosettle for about four hours at 37° C. The culture medium is aspiratedoff and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293cells are then washed with serum free medium, fresh medium is added andthe cells are incubated for about 5 days.

Approximately 24 hours after the transfections, the culture medium isremoved and replaced with culture medium (alone) or culture mediumcontaining 200 μCi/ml ³⁵S-cysteine and 200 μCi/ml ³⁵S-methionine. Aftera 12 hour incubation, the conditioned medium is collected, concentratedon a spin filter, and loaded onto a 15% SDS gel. The processed gel maybe dried and exposed to film for a selected period of time to reveal thepresence of PRO polypeptide. The cultures containing transfected cellsmay undergo further incubation (in serum free medium) and the medium istested in selected bioassays.

In an alternative technique, PRO polypeptide may be introduced into 293cells transiently using the dextran sulfate method described bySomparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells aregrown to maximal density in a spinner flask and 700 μg pRK5-PROpolypeptide DNA is added. The cells are first concentrated from thespinner flask by centrifugation and washed with PBS. The DNA-dextranprecipitate is incubated on the cell pellet for four hours. The cellsare treated with 20% glycerol for 90 seconds, washed with tissue culturemedium, and re-introduced into the spinner flask containing tissueculture medium, 5 μg/ml bovine insulin and 0.1 μg/ml bovine transferrin.After about four days, the conditioned media is centrifuged and filteredto remove cells and debris. The sample containing expressed PROpolypeptide can then be concentrated and purified by any selectedmethod, such as dialysis and/or column chromatography.

In another embodiment, PRO polypeptides can be expressed in CHO cells.The pRK5-PRO polypeptide can be transfected into CHO cells using knownreagents such as CaPO₄ or DEAE-dextran. As described above, the cellcultures can be incubated, and the medium replaced with culture medium(alone) or medium containing a radiolabel such as ³⁵S-methionine. Afterdetermining the presence of PRO polypeptide, the culture medium may bereplaced with serum free medium. Preferably, the cultures are incubatedfor about 6 days, and then the conditioned medium is harvested. Themedium containing the expressed PRO polypeptide can then be concentratedand purified by any selected method.

Epitope-tagged PRO polypeptide may also be expressed in host CHO cells.The PRO polypeptide may be subcloned out of the pRK5 vector. Thesubclone insert can undergo PCR to fuse in frame with a selected epitopetag such as a poly-his tag into a Baculovirus expression vector. Thepoly-his tagged PRO polypeptide insert can then be subcloned into a SV40driven vector containing a selection marker such as DHFR for selectionof stable clones. Finally, the CHO cells can be transfected (asdescribed above) with the SV40 driven vector. Labeling may be performed,as described above, to verify expression. The culture medium containingthe expressed poly-His tagged PRO polypeptide can then be concentratedand purified by any selected method, such as by Ni²⁻-chelate affinitychromatography.

PRO211, PRO217, PRO230, PRO219, PRO245, PRO221, PRO258, PRO301, PRO224,PRO222, PRO234, PRO229, PRO223, PRO328 and PRO332 were successfullyexpressed in CHO cells by both a transient and a stable expressionprocedure. In addition, PRO232, PRO265, PRO246, PRO228, PRO227, PRO220,PRO266, PRO269, PRO287, PRO214, PRO231, PRO233, PRO238, PRO244, PRO235,PRO236, PRO262, PRO239, PRO257, PRO260, PRO263, PRO270, PRO271, PRO272,PRO294, PRO295, PRO293, PRO247, PRO303 and PRO268 were successfullytransiently expressed in CHO cells.

Stable expression in CHO cells was performed using the followingprocedure. The proteins were expressed as an IgG construct(immunoadhesin), in which the coding sequences for the soluble forms(e.g. extracellular domains) of the respective proteins were fused to anIgG1 constant region sequence containing the hinge, CH2 and CH2 domainsand/or is a poly-His tagged form.

Following PCR amplification, the respective DNAs were subcloned in a CHOexpression vector using standard techniques as described in Ausubel etal., Current Protocols of Molecular Biology, Unit 3.16, John Wiley andSons (1997). CHO expression vectors are constructed to have compatiblerestriction sites 5′ and 3′ of the DNA of interest to allow theconvenient shuttling of cDNA's. The vector used expression in CHO cellsis as described in Lucas et al., Nucl. Acids Res. 24:9 (1774-1779(1996), and uses the SV40 early promoter/enhancer to drive expression ofthe cDNA of interest and dihydrofolate reductase (DHFR). DHFR expressionpermits selection for stable maintenance of the plasmid followingtransfection.

Twelve micrograms of the desired plasmid DNA were introduced intoapproximately 10 million CHO cells using commercially availabletransfection reagents Superfect® (Quiagen), Dosper® or Fugene®(Boehringer Mannheim). The cells were grown and described in Lucas etal., supra. Approximately 3×10⁻⁷ cells are frozen in an ampule forfurther growth and production as described below.

The ampules containing the plasmid DNA were thawed by placement intowater bath and mixed by vortexing. The contents were pipetted into acentrifuge tube containing 10 mLs of media and centrifuged at 1000 rpmfor 5 minutes. The supernatant was aspirated and the cells wereresuspended in 10 mL of selective media (0.2 μm filtered PS20 with 5%0.2 μm diafiltered fetal bovine serum). The cells were then aliquotedinto a 100 mL spinner containing 90 mL of selective media. After 1-2days, the cells were transferred into a 250 mL spinner filled with 150mL selective growth medium and incubated at 37° C. After another 2-3days, a 250 mL, 500 mL and 2000 mL spinners were seeded with 3×10⁵cells/mL. The cell media was exchanged with fresh media bycentrifugation and resuspension in production medium. Although anysuitable CHO media may be employed, a production medium described inU.S. Pat. No. 5,122,469, issued Jun. 16, 1992 was actually used. 3 Lproduction spinner is seeded at 1.2×10⁶ cells/mL. On day 0, the cellnumber pH were determined. On day 1, the spinner was sampled andsparging with filtered air was commenced. On day 2, the spinner wassampled, the temperature shifted to 33° C., and 30 mL of 500 g/L glucoseand 0.6 mL of 10% antifoam (e.g., 35% polydimethylsiloxane emulsion, DowCorning 365 Medical Grade Emulsion). Throughout the production, pH wasadjusted as necessary to keep at around 7.2. After 10 days, or untilviability dropped below 70%, the cell culture was harvested bycentrifugtion and filtering through a 0.22 μm filter. The filtrate waseither stored at 4° C. or immediately loaded onto columns forpurification.

For the poly-His tagged constructs, the proteins were purified using aNi-NTA column (Qiagen). Before purification, imidazole was added to theconditioned media to a concentration of 5 mM. The conditioned media waspumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4,buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5ml/min. at 4° C. After loading, the column was washed with additionalequilibration buffer and the protein eluted with equilibration buffercontaining 0.25 M imidazole. The highly purified protein wassubsequently desalted into a storage buffer containing 10 mM Hepes, 0.14M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia)column and stored at −80° C.

Immunoadhesin (Fc containing) constructs of were purified from theconditioned media as follows. The conditioned medium was pumped onto a 5ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Naphosphate buffer, pH 6.8. After loading, the column was washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein was immediately neutralized bycollecting 1 ml fractions into tubes containing 275 μL of 1 M Trisbuffer, pH 9. The highly purified protein was subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity was assessed by SDS polyacrylamide gels and by N-terminalamino acid sequencing by Edman degradation.

PRO211, PRO217, PRO230, PRO232, PRO187, PRO265, PRO219, PRO246, PRO228,PRO533, PRO245, PRO221, PRO227, PRO220, PRO258, PRO266, PRO269, PRO287,PRO214, PRO317, PRO301, PRO224, PRO222, PRO234, PRO231, PRO229, PRO233,PRO238, PRO223, PRO235, PRO236, PRO262, PRO239, PRO257, PRO260, PRO263,PRO270, PRO271, PRO272, PRO294, PRO295, PRO293, PRO247, PRO304, PRO302,PRO307, PRO303, PRO343, PRO328, PRO326, PRO331, PRO332, PRO334, PRO346,PRO268, PRO330, PRO310 and PRO339 were also successfully transientlyexpressed in COS cells.

Example 55 Expression of PRO Polypeptides in Yeast

The following method describes recombinant expression of a desired PROpolypeptide in yeast.

First, yeast expression vectors are constructed for intracellularproduction or secretion of PRO polypeptides from the ADH2/GAPDHpromoter. DNA encoding a desired PRO polypeptide, a selected signalpeptide and the promoter is inserted into suitable restriction enzymesites in the selected plasmid to direct intracellular expression of thePRO polypeptide. For secretion, DNA encoding the PRO polypeptide can becloned into the selected plasmid, together with DNA encoding theADH2/GAPDH promoter, the yeast alpha-factor secretory signal/leadersequence, and linker sequences (if needed) for expression of the PROpolypeptide.

Yeast cells, such as yeast strain AB 110, can then be transformed withthe expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

Recombinant PRO polypeptide can subsequently be isolated and purified byremoving the yeast cells from the fermentation medium by centrifugationand then concentrating the medium using selected cartridge filters. Theconcentrate containing the PRO polypeptide may further be purified usingselected column chromatography resins.

Example 56 Expression of PRO Polypeptides in Baculovirus-Infected InsectCells

The following method describes recombinant expression of PROpolypeptides in Baculovirus-infected insect cells.

The desired PRO polypeptide is fused upstream of an epitope tagcontained with a baculovirus expression vector. Such epitope tagsinclude poly-his tags and immunoglobulin tags (like Fc regions of IgG).A variety of plasmids may be employed, including plasmids derived fromcommercially available plasmids such as pVL1393 (Novagen). Briefly, thePRO polypeptide or the desired portion of the PRO polypeptide (such asthe sequence encoding the extracellular domain of a transmembraneprotein) is amplified by PCR with primers complementary to the 5′ and 3′regions. The 5′ primer may incorporate flanking (selected) restrictionenzyme sites. The product is then digested with those selectedrestriction enzymes and subcloned into the expression vector.

Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4-5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression is performed as described byO'Reilley et al., Baculovirus expression vectors: A laboratory Manual,Oxford: Oxford University Press (1994).

Expressed poly-his tagged PRO polypeptide can then be purified, forexample, by Ni²⁺-chelate affinity chromatography as follows. Extractsare prepared from recombinant virus-infected Sf9 cells as described byRupert et al., Nature, 362:175-179 (1993). Briefly, Sf9 cells arewashed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mMMgCl₂; 0.1 mM EDTA; 10% Glycerol; 0.1% NP-40; 0.4 M KCl), and sonicatedtwice for 20 seconds on ice. The sonicates are cleared bycentrifugation, and the supernatant is diluted 50-fold in loading buffer(50 mM phosphate, 300 mM NaCl, 10% Glycerol, pH 7.8) and filteredthrough a 0.45 μm filter. A Ni²⁺-NTA agarose column (commerciallyavailable from Qiagen) is prepared with a bed volume of 5 mL, washedwith 25 mL of water and equilibrated with 25 mL of loading buffer. Thefiltered cell extract is loaded onto the column at 0.5 mL per minute.The column is washed to baseline A₂₈₀ with loading buffer, at whichpoint fraction collection is started. Next, the column is washed with asecondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% Glycerol, pH6.0), which elutes nonspecifically bound protein. After reaching A₂₈₀baseline again, the column is developed with a 0 to 500 mM Imidazolegradient in the secondary wash buffer. One mL fractions are collectedand analyzed by SDS-PAGE and silver staining or western blot withNi²⁺-NTA-conjugated to alkaline phosphatase (Qiagen). Fractionscontaining the eluted His₁₀-tagged PRO polypeptide are pooled anddialyzed against loading buffer.

Alternatively, purification of the IgG tagged (or Fc tagged) PROpolypeptide can be performed using known chromatography techniques,including for instance, Protein A or protein G column chromatography.

PRO211, PRO217, PRO230, PRO187, PRO265, PRO246, PRO228, PRO533, PRO245,PRO221, PRO220, PRO258, PRO266, PRO269, PRO287, PRO214, PRO301, PRO224,PRO222, PRO234, PRO231, PRO229, PRO235, PRO239, PRO257, PRO272, PRO294,PRO295, PRO328, PRO326, PRO331, PRO334, PRO346 and PRO310 weresuccessfully expressed in baculovirus infected Sf9 or high5 insectcells. While the expression was actually performed in a 0.5-2 L scale,it can be readily scaled up for larger (e.g. 8 L) preparations. Theproteins were expressed as an IgG construct (immunoadhesin), in whichthe protein extracellular region was fused to an IgG1 constant regionsequence containing the hinge, CH2 and CH3 domains and/or in poly-Histagged forms.

Following PCR amplification, the respective coding sequences weresubcloned into a baculovirus expression vector (pb.PH.IgG for IgGfusions and pb.PH.His.c for poly-His tagged proteins), and the vectorand Baculogold® baculovirus DNA (Pharmingen) were co-transfected into105 Spodoptera frugiperda (“Sf9”) cells (ATCC CRL 1711), usingLipofectin (Gibco BRL). pb.PH.IgG and pb.PH.His are modifications of thecommercially available baculovirus expression vector pVL1393(Pharmingen), with modified polylinker regions to include the His or Fctag sequences. The cells were grown in Hink's TNM-FH medium supplementedwith 10% FBS (Hyclone). Cells were incubated for 5 days at 28° C. Thesupernatant was harvested and subsequently used for the first viralamplification by infecting Sf9 cells in Hink's TNM-FH mediumsupplemented with 10% FBS at an approximate multiplicity of infection(MO1) of 10. Cells were incubated for 3 days at 28° C. The supernatantwas harvested and the expression of the constructs in the baculovirusexpression vector was determined by batch binding of 1 ml of supernatantto 25 mL of Ni-NTA beads (QIAGEN) for histidine tagged proteins orProtein-A Sepharose CL-4B beads (Pharmacia) for IgG tagged proteinsfollowed by SDS-PAGE analysis comparing to a known concentration ofprotein standard by Coomassie blue staining.

The first viral amplification supernatant was used to infect a spinnerculture (500 ml) of Sf9 cells grown in ESF-921 medium (ExpressionSystems LLC) at an approximate MOI of 0.1. Cells were incubated for 3days at 28° C. The supernatant was harvested and filtered. Batch bindingand SDS-PAGE analysis was repeated, as necessary, until expression ofthe spinner culture was confirmed.

The conditioned medium from the transfected cells (0.5 to 3 L) washarvested by centrifugation to remove the cells and filtered through0.22 micron filters. For the poly-His tagged constructs, the proteinconstruct were purified using a Ni-NTA column (Qiagen). Beforepurification, imidazole was added to the conditioned media to aconcentration of 5 mM. The conditioned media were pumped onto a 6 mlNi-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4° C. Afterloading, the column was washed with additional equilibration buffer andthe protein eluted with equilibration buffer containing 0.25 Mimidazole. The highly purified protein was subsequently desalted into astorage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at −80° C.

Immunoadhesin (Fc containing) constructs of proteins were purified fromthe conditioned media as follows. The conditioned media were pumped ontoa 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mMNa phosphate buffer, pH 6.8. After loading, the column was washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein was immediately neutralized bycollecting 1 ml fractions into tubes containing 275 mL of 1 M Trisbuffer, pH 9. The highly purified protein was subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity of the proteins was verified by SDS polyacrylamide gel (PEG)electrophoresis and N-terminal amino acid sequencing by Edmandegradation.

Example 57 Preparation of Antibodies that Bind to PRO Polypeptides

This example illustrates preparation of monoclonal antibodies which canspecifically bind to a PRO polypeptide.

Techniques for producing the monoclonal antibodies are known in the artand are described, for instance, in Goding, supra. Immunogens that maybe employed include purified PRO polypeptide, fusion proteins containingthe PRO polypeptide, and cells expressing recombinant PRO polypeptide onthe cell surface. Selection of the immunogen can be made by the skilledartisan without undue experimentation.

Mice, such as Balb/c, are immunized with the PRO polypeptide immunogenemulsified in complete Freund's adjuvant and injected subcutaneously orintraperitoneally in an amount from 1-100 micrograms. Alternatively, theimmunogen is emulsified in MPL-TDM adjuvant (Ribi ImmunochemicalResearch, Hamilton, Mont.) and injected into the animal's hind footpads. The immunized mice are then boosted 10 to 12 days later withadditional immunogen emulsified in the selected adjuvant. Thereafter,for several weeks, the mice may also be boosted with additionalimmunization injections. Serum samples may be periodically obtained fromthe mice by retro-orbital bleeding for testing in ELISA assays to detectanti-PRO polypeptide antibodies.

After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of PRO polypeptide. Three to four days later, the mice aresacrificed and the spleen cells are harvested. The spleen cells are thenfused (using 35% polyethylene glycol) to a selected murine myeloma cellline such as P3×63AgU.1, available from ATCC, No. CRL 1597. The fusionsgenerate hybridoma cells which can then be plated in 96 well tissueculture plates containing HAT (hypoxanthine, aminopterin, and thymidine)medium to inhibit proliferation of non-fused cells, myeloma hybrids, andspleen cell hybrids.

The hybridoma cells will be screened in an ELISA for reactivity againstthe PRO polypeptide. Determination of “positive” hybridoma cellssecreting the desired monoclonal antibodies against the PRO polypeptideis within the skill in the art.

The positive hybridoma cells can be injected intraperitoneally intosyngeneic Balb/c mice to produce ascites containing the anti-PROpolypeptide monoclonal antibodies. Alternatively, the hybridoma cellscan be grown in tissue culture flasks or roller bottles. Purification ofthe monoclonal antibodies produced in the ascites can be accomplishedusing ammonium sulfate precipitation, followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can be employed.

Example 58 Chimeric PRO Polypeptides

PRO polypeptides may be expressed as chimeric proteins with one or moreadditional polypeptide domains added to facilitate protein purification.Such purification facilitating domains include, but are not limited to,metal chelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals, protein A domains that allowpurification on immobilized immunoglobulin, and the domain utilized inthe FLAGS™ extension/affinity purification system (Immunex Corp.,Seattle Wash.). The inclusion of a cleavable linker sequence such asFactor XA or enterokinase (Invitrogen, San Diego Calif.) between thepurification domain and the PRO polypeptide sequence may be useful tofacilitate expression of DNA encoding the PRO polypeptide.

Example 59 Purification of PRO Polypeptides Using Specific Antibodies

Native or recombinant PRO polypeptides may be purified by a variety ofstandard techniques in the art of protein purification. For example,pro-PRO polypeptide, mature PRO polypeptide, or pre-PRO polypeptide ispurified by immunoaffinity chromatography using antibodies specific forthe PRO polypeptide of interest. In general, an immunoaffinity column isconstructed by covalently coupling the anti-PRO polypeptide antibody toan activated chromatographic resin.

Polyclonal immunoglobulins are prepared from immune sera either byprecipitation with ammonium sulfate or by purification on immobilizedProtein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise,monoclonal antibodies are prepared from mouse ascites fluid by ammoniumsulfate precipitation or chromatography on immobilized Protein A.Partially purified immunoglobulin is covalently attached to achromatographic resin such as CnBr-activated SEPHAROSE™ (Pharmacia LKBBiotechnology). The antibody is coupled to the resin, the resin isblocked, and the derivative resin is washed according to themanufacturer's instructions.

Such an immunoaffinity column is utilized in the purification of PROpolypeptide by preparing a fraction from cells containing PROpolypeptide in a soluble form. This preparation is derived bysolubilization of the whole cell or of a subcellular fraction obtainedvia differential centrifugation by the addition of detergent or by othermethods well known in the art. Alternatively, soluble PRO polypeptidecontaining a signal sequence may be secreted in useful quantity into themedium in which the cells are grown.

A soluble PRO polypeptide-containing preparation is passed over theimmunoaffinity column, and the column is washed under conditions thatallow the preferential absorbance of PRO polypeptide (e.g., high ionicstrength buffers in the presence of detergent). Then, the column iseluted under conditions that disrupt antibody/PRO polypeptide binding(e.g., a low pH buffer such as approximately pH 2-3, or a highconcentration of a chaotrope such as urea or thiocyanate ion), and PROpolypeptide is collected.

Example 60 Drug Screening

This invention is particularly useful for screening compounds by usingPRO polypeptides or binding fragment thereof in any of a variety of drugscreening techniques. The PRO polypeptide or fragment employed in such atest may either be free in solution, affixed to a solid support, borneon a cell surface, or located intracellularly. One method of drugscreening utilizes eukaryotic or prokaryotic host cells which are stablytransformed with recombinant nucleic acids expressing the PROpolypeptide or fragment. Drugs are screened against such transformedcells in competitive binding assays. Such cells, either in viable orfixed form, can be used for standard binding assays. One may measure,for example, the formation of complexes between PRO polypeptide or afragment and the agent being tested. Alternatively, one can examine thediminution in complex formation between the PRO polypeptide and itstarget cell or target receptors caused by the agent being tested.

Thus, the present invention provides methods of screening for drugs orany other agents which can affect a PRO polypeptide-associated diseaseor disorder. These methods comprise contacting such an agent with an PROpolypeptide or fragment thereof and assaying (I) for the presence of acomplex between the agent and the PRO polypeptide or fragment, or (ii)for the presence of a complex between the PRO polypeptide or fragmentand the cell, by methods well known in the art. In such competitivebinding assays, the PRO polypeptide or fragment is typically labeled.After suitable incubation, free PRO polypeptide or fragment is separatedfrom that present in bound form, and the amount of free or uncomplexedlabel is a measure of the ability of the particular agent to bind to PROpolypeptide or to interfere with the PRO polypeptide/cell complex.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to a polypeptide and isdescribed in detail in WO 84/03564, published on Sep. 13, 1984. Brieflystated, large numbers of different small peptide test compounds aresynthesized on a solid substrate, such as plastic pins or some othersurface. As applied to a PRO polypeptide, the peptide test compounds arereacted with PRO polypeptide and washed. Bound PRO polypeptide isdetected by methods well known in the art. Purified PRO polypeptide canalso be coated directly onto plates for use in the aforementioned drugscreening techniques. In addition, non-neutralizing antibodies can beused to capture the peptide and immobilize it on the solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding PROpolypeptide specifically compete with a test compound for binding to PROpolypeptide or fragments thereof. In this manner, the antibodies can beused to detect the presence of any peptide which shares one or moreantigenic determinants with PRO polypeptide.

Example 61 Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptide of interest (i.e., a PRO polypeptide) orof small molecules with which they interact, e.g., agonists,antagonists, or inhibitors. Any of these examples can be used to fashiondrugs which are more active or stable forms of the PRO polypeptide orwhich enhance or interfere with the function of the PRO polypeptide invivo (cf., Hodgson, Bio/Technology, 9: 19-21(1991)).

In one approach, the three-dimensional structure of the PRO polypeptide,or of an PRO polypeptide-inhibitor complex, is determined by x-raycrystallography, by computer modeling or, most typically, by acombination of the two approaches. Both the shape and charges of the PROpolypeptide must be ascertained to elucidate the structure and todetermine active site(s) of the molecule. Less often, useful informationregarding the structure of the PRO polypeptide may be gained by modelingbased on the structure of homologous proteins. In both cases, relevantstructural information is used to design analogous PRO polypeptide-likemolecules or to identify efficient inhibitors. Useful examples ofrational drug design may include molecules which have improved activityor stability as shown by Braxton and Wells, Biochemistry, 31:7796-7801(1992) or which act as inhibitors, agonists, or antagonists of nativepeptides as shown by Athauda et al., J. Biochem., 113:742-746 (1993).

It is also possible to isolate a target-specific antibody, selected byfunctional assay, as described above, and then to solve its crystalstructure. This approach, in principle, yields a pharmacore upon whichsubsequent drug design can be based. It is possible to bypass proteincrystallography altogether by generating anti-idiotypic antibodies(anti-ids) to a functional, pharmacologically active antibody. As amirror image of a mirror image, the binding site of the anti-ids wouldbe expected to be an analog of the original receptor. The anti-id couldthen be used to identify and isolate peptides from banks of chemicallyor biologically produced peptides. The isolated peptides would then actas the pharmacore.

By virtue of the present invention, sufficient amounts of the PROpolypeptide may be made available to perform such analytical studies asX-ray crystallography. In addition, knowledge of the PRO polypeptideamino acid sequence provided herein will provide guidance to thoseemploying computer modeling techniques in place of or in addition tox-ray crystallography.

Example 62 Diagnostic Test Using PRO317 Polypeptide-Specific Antibodies

Particular anti-PRO317 polypeptide antibodies are useful for thediagnosis of prepathologic conditions, and chronic or acute diseasessuch as gynecological diseases or ischemic diseases which arecharacterized by differences in the amount or distribution of PRO317.PRO317 has been found to be expressed in human kidney and is thus likelyto be associated with abnormalities or pathologies which affect thisorgan. Further, since it is so closely related to EBAF-1, it is likelyto affect the endometrium and other genital tissues. Further, due tolibrary sources of certain ESTs, it appears that PRO317 may be involvedas well in forming blood vessels and hence to be a modulator ofangiogenesis.

Diagnostic tests for PRO317 include methods utilizing the antibody and alabel to detect PRO317 in human body fluids, tissues, or extracts ofsuch tissues. The polypeptide and antibodies of the present inventionmay be used with or without modification. Frequently, the polypeptideand antibodies will be labeled by joining them, either covalently ornoncovalently, with a substance which provides for a detectable signal.A wide variety of labels and conjugation techniques are known and havebeen reported extensively in both the scientific and patent literature.Suitable labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent agents, chemiluminescent agents, magneticparticles, and the like. Patents teaching the use of such labels includeU.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149; and 4,366,241. Also, recombinant immunoglobulins may beproduced as shown in U.S. Pat. No. 4,816,567.

A variety of protocols for measuring soluble or membrane-bound PRo317,using either polyclonal or monoclonal antibodies specific for thatPRO317, are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), radioreceptor assay(RRA), and fluorescent activated cell sorting (FACS). A two-sitemonoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epitopes on PRO317 is preferred, but a competitivebinding assay may be employed. These assays are described, among otherplaces, in Maddox et al. J. Exp. Med., 158:1211 (1983).

Example 63 Identification of PRO317 Receptors

Purified PRO317 is useful for characterization and purification ofspecific cell surface receptors and other binding molecules. Cells whichrespond to PRO317 by metabolic changes or other specific responses arelikely to express a receptor for PRO317. Such receptors include, but arenot limited to, receptors associated with and activated by tyrosine andserine/threonine kinases. See Kolodziejczyk and Hall, supra, for areview on known receptors for the TGF-superfamily. Candidate receptorsfor this superfamily fall into two primary groups, termed type I andtype II receptors. Both types are serine/threonine kinases. Uponactivation by the appropriate ligand, type I and type II receptorsphysically interact to form hetero-oligomers and subsequently activateintracellular signaling cascades, ultimately regulating genetranscription and expression. In addition, TGF-binds to a third receptorclass, type III, a membrane-anchored proteoglycan lacking the kinaseactivity typical of signal transducing molecules.

PRO317 receptors or other PRO317-binding molecules may be identified byinteraction with radiolabeled PRO317. Radioactive labels may beincorporated into PRO317 by various methods known in the art. Apreferred embodiment is the labeling of primary amino groups in PRO317with ¹²⁵I Bolton-Hunter reagent (Bolton and Hunter, Biochem. J., 133:529(1973)), which has been used to label other polypeptides withoutconcomitant loss of biological activity (Hebert et al., J. Biol. Chem.,266:18989 (1991); McColl et al., J. Immunol., 150:4550-4555 (1993)).Receptor-bearing cells are incubated with labeled PRO317. The cells arethen washed to removed unbound PRO317, and receptor-bound PRO317 isquantified. The data obtained using different concentrations of PRO317are used to calculate values for the number and affinity of receptors.

Labeled PRO317 is useful as a reagent for purification of its specificreceptor. In one embodiment of affinity purification, PRO317 iscovalently coupled to a chromatography column. Receptor-bearing cellsare extracted, and the extract is passed over the column. The receptorbinds to the column by virtue of its biological affinity for PRO317. Thereceptor is recovered from the column and subjected to N-terminalprotein sequencing. This amino acid sequence is then used to designdegenerate oligonucleotide probes for cloning the receptor gene.

In an alternative method, mRNA is obtained from receptor-bearing cellsand made into a cDNA library. The library is transfected into apopulation of cells, and those cells expressing the receptor areselected using fluorescently labeled PRO317. The receptor is identifiedby recovering and sequencing recombinant DNA from highly labeled cells.

In another alternative method, antibodies are raised against the surfaceof receptor bearing cells, specifically monoclonal antibodies. Themonoclonal antibodies are screened to identify those which inhibit thebinding of labeled PRO317. These monoclonal antibodies are then used inaffinity purification or expression cloning of the receptor.

Soluble receptors or other soluble binding molecules are identified in asimilar manner. Labeled PRO317 is incubated with extracts or otherappropriate materials derived from the uterus. After incubation, PRO317complexes larger than the size of purified PRO317 are identified by asizing technique such as size-exclusion chromatography or densitygradient centrifugation and are purified by methods known in the art.The soluble receptors or binding protein(s) are subjected to N-terminalsequencing to obtain information sufficient for database identification,if the soluble protein is known, or for cloning, if the soluble proteinis unknown.

Example 64 Determination of PRO317-Induced Cellular Response

The biological activity of PRO317 is measured, for example, by bindingof an PRO317 of the invention to an PRO317 receptor. A test compound isscreened as an antagonist for its ability to block binding of PRO317 tothe receptor. A test compound is screened as an agonist of the PRO317for its ability to bind an PRO317 receptor and influence the samephysiological events as PRO317 using, for example, the KIRA-ELISA assaydescribed by Sadick et al., Analytical Biochemistry, 235:207-214 (1996)in which activation of a receptor tyrosine kinase is monitored byimmuno-capture of the activated receptor and quantitation of the levelof ligand-induced phosphorylation. The assay may be adapted to monitorPRO317-induced receptor activation through the use of an PRO317receptor-specific antibody to capture the activated receptor. Thesetechniques are also applicable to other PRO polypeptides describedherein.

Example 65 Use of PRO224 for Screening Compounds

PRO224 is expressed in a cell stripped of membrane proteins and capableof expressing PRO224. Low density lipoproteins having a detectable labelare added to the cells and incubated for a sufficient time forendocytosis. The cells are washed. The cells are then analysed for labelbound to the membrane and within the cell after cell lysis. Detection ofthe low density lipoproteins within the cell determines that PRO224 iswithin the family of low density lipoprotein receptor proteins. Membersfound within this family are then used for screening compounds whichaffect these receptors, and particularly the uptake of cholesterol viathese receptors.

Example 66 Ability of PRO Polypeptides to Inhibit Vascular EndothelialGrowth Factor (VEGF) Stimulated Proliferation of Endothelial Cell Growth(Assay 9)

The ability of various PRO polypeptides to inhibit VEGF stimulatedproliferation of endothelial cells was tested. Polypeptides testingpositive in this assay are useful for inhibiting endothelial cell growthin mammals where such an effect would be beneficial, e.g., forinhibiting tumor growth.

Specifically, bovine adrenal cortical capillary endothelial cells (ACE)(from primary culture, maximum of 12-14 passages) were plated in 96-wellplates at 500 cells/well per 100 microliter. Assay media included lowglucose DMEM, 10% calf serum, 2 mM glutamine, and 1×penicillin/streptomycin/fungizone. Control wells included the following:(1) no ACE cells added; (2) ACE cells alone; (3) ACE cells plus 5 ng/mlFGF; (4) ACE cells plus 3 ng/ml VEGF; (5) ACE cells plus 3 ng/ml VEGFplus 1 ng/ml TGF-beta; and (6) ACE cells plus 3 ng/ml VEGF plus 5 ng/mlLIF. The test samples, poly-his tagged PRO polypeptides (in 100microliter volumes), were then added to the wells (at dilutions of 1%,0.1% and 0.01%, respectively). The cell cultures were incubated for 6-7days at 37° C./5% CO₂. After the incubation, the media in the wells wasaspirated, and the cells were washed 1× with PBS. An acid phosphatasereaction mixture (100 microliter; 0.1M sodium acetate, pH 5.5, 0.1%Triton X-100, 10 mM p-nitrophenyl phosphate) was then added to eachwell. After a 2 hour incubation at 37° C., the reaction was stopped byaddition of 10 microliters 1N NaOH. Optical density (OD) was measured ona microplate reader at 405 nm.

The activity of PRO polypeptides was calculated as the percentinhibition of VEGF (3 ng/ml) stimulated proliferation (as determined bymeasuring acid phosphatase activity at OD 405 nm) relative to the cellswithout stimulation. TGF-beta was employed as an activity reference at 1ng/ml, since TGF-beta blocks 70-90% of VEGF-stimulated ACE cellproliferation. The results are indicative of the utility of the PROpolypeptides in cancer therapy and specifically in inhibiting tumorangiogenesis. Numerical values (relative inhibition) are determined bycalculating the percent inhibition of VEGF stimulated proliferation bythe PRO polypeptides relative to cells without stimulation and thendividing that percentage into the percent inhibition obtained by TGF-βat 1 ng/ml which is known to block 70-90% of VEGF stimulated cellproliferation. The results are considered positive if the PROpolypeptide exhibits 30% or greater inhibition of VEGF stimulation ofendothelial cell growth (relative inhibition 30% or greater).

The following polypeptides tested positive in this assay: PRO211,PRO217, PRO187, PRO219, PRO246, PRO228, PRO245, PRO221, PRO258, PRO301,PRO224, PRO272, PRO328, PRO331, PRO224, PRO328, PRO272, PRO301, PRO331and PRO214.

Example 67 Retinal Neuron Survival (Assay 52)

This example demonstrates that certain PRO polypeptides have efficacy inenhancing the survival of retinal neuron cells and, therefore, areuseful for the therapeutic treatment of retinal disorders or injuriesincluding, for example, treating sight loss in mammals due to retinitispigmentosum, AMD, etc.

Sprague Dawley rat pups at postnatal day 7 (mixed population: glia andretinal neuronal types) are killed by decapitation following CO₂anesthesia and the eyes are removed under sterile conditions. The neuralretina is dissected away from the pigment epithelium and other oculartissue and then dissociated into a single cell suspension using 0.25%trypsin in Ca²⁺, Mg²⁺-free PBS. The retinas are incubated at 37° C. for7-10 minutes after which the trypsin is inactivated by adding 1 mlsoybean trypsin inhibitor. The cells are plated at 100,000 cells perwell in 96 well plates in DMEM/F12 supplemented with N2 and with orwithout the specific test PRO polypeptide. Cells for all experiments aregrown at 37° C. in a water saturated atmosphere of 5% CO₂. After 2-3days in culture, cells are stained with calcein AM then fixed using 4%paraformaldehyde and stained with DAPI for determination of total cellcount. The total cells (fluorescent) are quantified at 20× objectivemagnification using CCD camera and NIH image software for MacIntosh.Fields in the well are chosen at random.

The effect of various concentration of PRO polypeptides are reportedherein where percent survival is calculated by dividing the total numberof calcein AM positive cells at 2-3 days in culture by the total numberof DAPI-labeled cells at 2-3 days in culture. Anything above 30%survival is considered positive.

The following PRO polypeptides tested positive in this assay usingpolypeptide concentrations within the range of 0.01% to 1.0% in theassay: PRO220 and PRO346.

Example 68 Rod Photoreceptor Cell Survival (Assay 56)

This assay shows that certain polypeptides of the invention act toenhance the survival/proliferation of rod photoreceptor cells and,therefore, are useful for the therapeutic treatment of retinal disordersor injuries including, for example, treating sight loss in mammals dueto retinitis pigmentosum, AMD, etc. Sprague Dawley rat pups at 7 daypostnatal (mixed population: glia and retinal neuronal cell types) arekilled by decapitation following CO₂ anesthesis and the eyes are removedunder sterile conditions. The neural retina is dissected away form thepigment epithelium and other ocular tissue and then dissociated into asingle cell suspension using 0.25% trypsin in Ca²⁺, Mg²⁺-free PBS. Theretinas are incubated at 37° C. for 7-10 minutes after which the trypsinis inactivated by adding 1 ml soybean trypsin inhibitor. The cells areplated at 100,000 cells per well in 96 well plates in DMEM/F12supplemented with N₂. Cells for all experiments are grown at 37° C. in awater saturated atmosphere of 5% CO₂. After 2-3 days in culture, cellsare fixed using 4% paraformaldehyde, and then stained using CellTrackerGreen CMFDA. Rho 4D2 (ascites or IgG 1:100), a monoclonal antibodydirected towards the visual pigment rhodopsin is used to detect rodphotoreceptor cells by indirect immunofluorescence. The results arecalculated as % survival: total number of calcein—rhodopsin positivecells at 2-3 days in culture, divided by the total number of rhodopsinpositive cells at time 2-3 days in culture. The total cells(fluorescent) are quantified at 20× objective magnification using a CCDcamera and NIH image software for MacIntosh. Fields in the well arechosen at random.

The following polypeptides tested positive in this assay: PRO220 andPRO346.

Example 69 Induction of Endothelial Cell Apoptosis (Assay 73)

The ability of PRO polypeptides to induce apoptosis in endothelial cellswas tested in human venous umbilical vein endothelial cells (HUVEC, CellSystems). A positive test in the assay is indicative of the usefulnessof the polypeptide in therapeutically treating tumors as well asvascular disorders where inducing apoptosis of endothelial cells wouldbe beneficial.

The cells were plated on 96-well microtiter plates (Amersham LifeScience, cytostar-T scintillating microplate, RPNQ160, sterile,tissue-culture treated, individually wrapped), in 10% serum (CSG-medium,Cell Systems), at a density of 2×10⁴ cells per well in a total volume of100 μl. On day 2, test samples containing the PRO polypeptide were addedin triplicate at dilutions of 1%, 0.33% and 0.11%. Wells without cellswere used as a blank and wells with cells only were used as a negativecontrol. As a positive control 1:3 serial dilutions of 50 μl of a 3×stock of staurosporine were used. The ability of the PRO polypeptide toinduce apoptosis was determined by processing of the 96 well plates fordetection of Annexin V, a member of the calcium and phospholipid bindingproteins, to detect apoptosis.

0.2 ml Annexin V—Biotin stock solution (100 μg/ml) was diluted in 4.6 ml2× Ca²⁺ binding buffer and 2.5% BSA (1:25 dilution). 50 μl of thediluted Annexin V—Biotin solution was added to each well (exceptcontrols) to a final concentration of 1.0 μg/ml. The samples wereincubated for 10-15 minutes with Annexin-Biotin prior to direct additionof ³⁵S-Streptavidin. ³⁵S-Streptavidin was diluted in 2× Ca²⁺ Bindingbuffer, 2.5% BSA and was added to all wells at a final concentration of3×10⁴ cpm/well. The plates were then sealed, centrifuged at 1000 rpm for15 minutes and placed on orbital shaker for 2 hours. The analysis wasperformed on a 1450 Microbeta Trilux (Wallac). Percent above backgroundrepresents the percentage amount of counts per minute above the negativecontrols. Percents greater than or equal to 30% above background areconsidered positive.

The following PRO polypeptides tested positive in this assay: PRO228,PRO217 and PRO301.

Example 70 PDB12 Cell Inhibition (Assay 40)

This example demonstrates that various PRO polypeptides have efficacy ininhibiting protein production by PDB12 pancreatic ductal cells and are,therefore, useful in the therapeutic treatment of disorders whichinvolve protein secretion by the pancreas, including diabetes, and thelike.

PDB12 pancreatic ductal cells are plated on fibronectin coated 96 wellplates at 1.5×10³ cells per well in 100 μL/180 μL of growth media. 100μL of growth media with the PRO polypeptide test sample or negativecontrol lacking the PRO polypeptide is then added to well, for a finalvolume of 200 μL. Controls contain growth medium containing a proteinshown to be inactive in this assay. Cells are incubated for 4 days at37° C. 20 μL of Alamar Blue Dye (AB) is then added to each well and theflourescent reading is measured at 4 hours post addition of AB, on amicrotiter plate reader at 530 μm excitation and 590 nm emission. Thestandard employed is cells without Bovine Pituitary Extract (BPE) andwith various concentrations of BPE. Buffer or CM controls from unknownsare run 2 times on each 96 well plate.

These assays allow one to calculate a percent decrease in proteinproduction by comparing the Alamar Blue Dye calculated proteinconcentration produced by the PRO polypeptide-treated cells with theAlamar Blue Dye calculated protein concentration produced by thenegative control cells. A percent decrease in protein production ofgreater than or equal to 25% as compared to the negative control cellsis considered positive.

The following polypeptides tested positive in this assay: PRO211,PRO287, PRO301 and PRO293.

Example 71 Stimulation of Adult Heart Hypertrophy (Assay 2)

This assay is designed to measure the ability of various PROpolypeptides to stimulate hypertrophy of adult heart. PRO polypeptidestesting positive in this assay would be expected to be useful for thetherapeutic treatment of various cardiac insufficiency disorders.

Ventricular myocytes freshly isolated from adult (250 g) Sprague Dawleyrats are plated at 2000 cell/well in 180 μl volume. Cells are isolatedand plated on day 1, the PRO polypeptide-containing test samples orgrowth medium only (negative control) (20 μl volume) is added on day 2and the cells are then fixed and stained on day 5. After staining, cellsize is visualized wherein cells showing no growth enhancement ascompared to control cells are given a value of 0.0, cells showing smallto moderate growth enhancement as compared to control cells are given avalue of 1.0 and cells showing large growth enhancement as compared tocontrol cells are given a value of 2.0. Any degree of growth enhancementas compared to the negative control cells is considered positive for theassay.

The following PRO polypeptides tested positive in this assay: PRO287,PRO301, PRO293 and PRO303.

Example 72 PDB12 Cell Proliferation (Assay 29)

This example demonstrates that various PRO polypeptides have efficacy ininducing proliferation of PDB 12 pancreatic ductal cells and are,therefore, useful in the therapeutic treatment of disorders whichinvolve protein secretion by the pancreas, including diabetes, and thelike.

PDB12 pancreatic ductal cells are plated on fibronectin coated 96 wellplates at 1.5×10³ cells per well in 100 μL/180 μL of growth media. 100μL of growth media with the PRO polypeptide test sample or negativecontrol lacking the PRO polypeptide is then added to well, for a finalvolume of 200 μL. Controls contain growth medium containing a proteinshown to be inactive in this assay. Cells are incubated for 4 days at37° C. 20 μL of Alamar Blue Dye (AB) is then added to each well and theflourescent reading is measured at 4 hours post addition of AB, on amicrotiter plate reader at 530 μm excitation and 590 nm emission. Thestandard employed is cells without Bovine Pituitary Extract (BPE) andwith various concentrations of BPE. Buffer or growth medium onlycontrols from unknowns are run 2 times on each 96 well plate.

Percent increase in protein production is calculated by comparing theAlamar Blue Dye calculated protein concentration produced by the PROpolypeptide-treated cells with the Alamar Blue Dye calculated proteinconcentration produced by the negative control cells. A percent increasein protein production of greater than or equal to 25% as compared to thenegative control cells is considered positive.

The following PRO polypeptides tested positive in this assay: PRO301 andPRO303.

Example 73 Enhancement of Heart Neonatal Hypertrophy (Assay 1)

This assay is designed to measure the ability of PRO polypeptides tostimulate hypertrophy of neonatal heart. PRO polypeptides testingpositive in this assay are expected to be useful for the therapeutictreatment of various cardiac insufficiency disorders.

Cardiac myocytes from 1-day old Harlan Sprague Dawley rats wereobtained. Cells (180 μl at 7.5×10⁴/ml, serum<0.1%, freshly isolated) areadded on day 1 to 96-well plates previously coated with DMEM/F12+4% FCS.Test samples containing the test PRO polypeptide or growth medium only(hegative control) (20 μl/well) are added directly to the wells onday 1. PGF (20 μl/well) is then added on day 2 at final concentration of10⁻⁶ M. The cells are then stained on day 4 and visually scored on day5, wherein cells showing no increase in size as compared to negativecontrols are scored 0.0, cells showing a small to moderate increase insize as compared to negative controls are scored 1.0 and cells showing alarge increase in size as compared to negative controls are scored 2.0.A positive result in the assay is a score of 1.0 or greater.

The following polypeptides tested positive in this assay: PRO224 andPRO231.

Example 74 Stimulatory Activity in Mixed Lymphocyte Reaction (MLR) Assay(Assay 24)

This example shows that certain polypeptides of the invention are activeas a stimulator of the proliferation of stimulated T-lymphocytes.Compounds which stimulate proliferation of lymphocytes are usefultherapeutically where enhancement of an immune response is beneficial. Atherapeutic agent may take the form of antagonists of the polypeptide ofthe invention, for example, murine-human chimeric, humanized or humanantibodies against the polypeptide.

The basic protocol for this assay is described in Current Protocols inImmunology, unit 3.12; edited by J E Coligan, A M Kruisbeek, D HMarglies, E M Shevach, W Strober, National Insitutes of Health,Published by John Wiley & Sons, Inc.

More specifically, in one assay variant, peripheral blood mononuclearcells (PBMC) are isolated from mammalian individuals, for example ahuman volunteer, by leukopheresis (one donor will supply stimulatorPBMCs, the other donor will supply responder PBMCs). If desired, thecells are frozen in fetal bovine serum and DMSO after isolation. Frozencells may be thawed overnight in assay media (37° C., 5% CO₂) and thenwashed and resuspended to 3×10⁶ cells/ml of assay media (RPMI; 10% fetalbovine serum, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES, 1%non-essential amino acids, 1% pyruvate). The stimulator PBMCs areprepared by irradiating the cells (about 3000 Rads).

The assay is prepared by plating in triplicate wells a mixture of:

-   -   100:1 of test sample diluted to 1% or to 0.1%,    -   50:1 of irradiated stimulator cells, and    -   50:1 of responder PBMC cells.        100 microliters of cell culture media or 100 microliter of        CD4-IgG is used as the control. The wells are then incubated at        37° C., 5% CO₂ for 4 days. On day 5, each well is pulsed with        tritiated thymidine (1.0 mC/well; Amersham). After 6 hours the        cells are washed 3 times and then the uptake of the label is        evaluated.

In another variant of this assay, PBMCs are isolated from the spleens ofBalb/c mice and C57B6 mice. The cells are teased from freshly harvestedspleens in assay media (RPMI; 10% fetal bovine serum, 1%penicillin/streptomycin, 1% glutamine, 1% HEPES, 1% non-essential aminoacids, 1% pyruvate) and the PBMCs are isolated by overlaying these cellsover Lympholyte M (Organon Teknika), centrifuging at 2000 rpm for 20minutes, collecting and washing the mononuclear cell layer in assaymedia and resuspending the cells to 1×10⁷ cells/ml of assay media. Theassay is then conducted as described above.

Positive increases over control are considered positive with increasesof greater than or equal to 180% being preferred. However, any valuegreater than control indicates a stimulatory effect for the testprotein.

The following PRO polypeptides tested positive in this assay: PRO245,PRO269, PRO217, PRO301, PRO266, PRO335, PRO331, PRO533 and PRO326.

Example 75 Pericyte c-Fos Induction (Assay 93)

This assay shows that certain polypeptides of the invention act toinduce the expression of c-fos in pericyte cells and, therefore, areuseful not only as diagnostic markers for particular types ofpericyte-associated tumors but also for giving rise to antagonists whichwould be expected to be useful for the therapeutic treatment ofpericyte-associated tumors. Specifically, on day 1, pericytes arereceived from VEC Technologies and all but 5 ml of media is removed fromflask. On day 2, the pericytes are trypsinized, washed, spun and thenplated onto 96 well plates. On day 7, the media is removed and thepericytes are treated with 100 μl of PRO polypeptide test samples andcontrols (positive control=DME+5% serum+/−PDGF at 500 ng/ml; negativecontrol=protein 32). Replicates are averaged and SD/CV are determined.Fold increase over Protein 32 (buffer control) value indicated bychemiluminescence units (RLU) luminometer reading verses frequency isplotted on a histogram. Two-fold above Protein 32 value is consideredpositive for the assay. ASY Matrix: Growth media=low glucose DMEM=20%FBS+1× pen strep+1× fungizone. Assay Media=low glucose DMEM+5% FBS.

The following polypeptides tested positive in this assay: PRO214,PRO219, PRO221 and PRO224.

Example 76 Ability of PRO Polypeptides to Stimulate the Release ofProteoglycans from Cartilage (Assay 97)

The ability of various PRO polypeptides to stimulate the release ofproteoglycans from cartilage tissue was tested as follows.

The metacarphophalangeal joint of 4-6 month old pigs was asepticallydissected, and articular cartilage was removed by free hand slicingbeing careful to avoid the underlying bone. The cartilage was minced andcultured in bulk for 24 hours in a humidified atmosphere of 95% air, 5%CO₂ in serum free (SF) media (DME/F12 1:1) woth 0.1% BSA and 100 U/mlpenicillin and 100 μg/ml streptomycin. After washing three times,approximately 100 mg of articular cartilage was aliquoted into micronicstubes and incubated for an additional 24 hours in the above SF media.PRO polypeptides were then added at 1% either alone or in combinationwith 18 ng/ml interleukin-1α, a known stimulator of proteoglycan releasefrom cartilage tissue. The supernatant was then harvested and assayedfor the amount of proteoglycans using the 1,9-dimethyl-methylene blue(DMB) colorimetric assay (Farndale and Buttle, Biochem. Biophys. Acta883:173-177 (1985)). A positive result in this assay indicates that thetest polypeptide will find use, for example, in the treatment ofsports-related joint problems, articular cartilage defects,osteoarthritis or rheumatoid arthritis.

When various PRO polypeptides were tested in the above assay, thepolypeptides demonstrated a marked ability to stimulate release ofproteoglycans from cartilage tissue both basally and after stimulationwith interleukin-1α and at 24 and 72 hours after treatment, therebyindicating that these PRO polypeptides are useful for stimulatingproteoglycan release from cartilage tissue. As such, these PROpolypeptides are useful for the treatment of sports-related jointproblems, articular cartilage defects, osteoarthritis or rheumatoidarthritis. The polypeptides testing positive in this assay are: PRO211.

Example 77 Skin Vascular Permeability Assay (Assay 64)

This assay shows that certain polypeptides of the invention stimulate animmune response and induce inflammation by inducing mononuclear cell,eosinophil and PMN infiltration at the site of injection of the animal.Compounds which stimulate an immune response are useful therapeuticallywhere stimulation of an immune response is beneficial. This skinvascular permeability assay is conducted as follows. Hairless guineapigs weighing 350 grams or more are anesthetized with ketamine (75-80mg/Kg) and 5 mg/Kg xylazine intramuscularly (IM). A sample of purifiedpolypeptide of the invention or a conditioned media test sample isinjected intradermally onto the backs of the test animals with 100 μlper injection site. It is possible to have about 10-30, preferably about16-24, injection sites per animal. One μl of Evans blue dye (1% inphysiologic buffered saline) is injected intracardially. Blemishes atthe injection sites are then measured (mm diameter) at 1 hr and 6 hrpost injection. Animals were sacrificed at 6 hrs after injection. Eachskin injection site is biopsied and fixed in formalin. The skins arethen prepared for histopathologic evaluation. Each site is evaluated forinflammatory cell infiltration into the skin. Sites with visibleinflammatory cell inflammation are scored as positive. Inflammatorycells may be neutrophilic, eosinophilic, monocytic or lymphocytic. Atleast a minimal perivascular infiltrate at the injection site is scoredas positive, no infiltrate at the site of injection is scored asnegative.

The following polypeptides tested positive in this assay: PRO245,PRO217, PRO326, PRO266, PRO272, PRO301, PRO331 and PRO335.

Example 78 Enhancement of Heart Neonatal Hypertrophy Induced by F2a(Assay 37)

This assay is designed to measure the ability of PRO polypeptides tostimulate hypertrophy of neonatal heart. PRO polypeptides testingpositive in this assay are expected to be useful for the therapeutictreatment of various cardiac insufficiency disorders.

Cardiac myocytes from 1-day old Harlan Sprague Dawley rats wereobtained. Cells (180 μl at 7.5×10⁴/ml, serum<0.1%, freshly isolated) areadded on day 1 to 96-well plates previously coated with DMEM/F12+4% FCS.Test samples containing the test PRO polypeptide (20 μl/well) are addeddirectly to the wells on day 1. PGF (20 μl/well) is then added on day 2at a final concentration of 10-6 M. The cells are then stained on day 4and visually scored on day 5. Visual scores are based on cell size,wherein cells showing no increase in size as compared to negativecontrols are scored 0.0, cells showing a small to moderate increase insize as compared to negative controls are scored 1.0 and cells showing alarge increase in size as compared to negative controls are scored 2.0.A score of 1.0 or greater is considered positive.

No PBS is included, since calcium concentration is critical for assayresponse. Plates are coated with DMEM/F12 plus 4% FCS (200 μl/well).Assay media included: DMEM/F12 (with 2.44 gm bicarbonate), 10 μg/mltransferrin, 1 μg/ml insulin, 1 μg/ml aprotinin, 2 mmol/L glutamine, 100U/ml penicillin G, 100 μg/ml streptomycin. Protein buffer containingmannitol (4%) gave a positive signal (score 3.5) at {fraction (1/10)}(0.4%) and {fraction (1/100)} (0.04%), but not at {fraction (1/1000)}(0.004%). Therefore the test sample buffer containing mannitol is notrun.

The following PRO polypeptides tested positive in this assay: PRO224.

Example 79 Inhibitory Activity in Mixed Lymphocyte Reaction (MLR) Assay(Assay 67)

This example shows that one or more of the polypeptides of the inventionare active as inhibitors of the proliferation of stimulatedT-lymphocytes. Compounds which inhibit proliferation of lymphocytes areuseful therapeutically where suppression of an immune response isbeneficial.

The basic protocol for this assay is described in Current Protocols inImmunology, unit 3.12; edited by J E Coligan, A M Kruisbeek, D HMarglies, E M Shevach, W Strober, National Insitutes of Health,Published by John Wiley & Sons, Inc.

More specifically, in one assay variant, peripheral blood mononuclearcells (PBMC) are isolated from mammalian individuals, for example ahuman volunteer, by leukopheresis (one donor will supply stimulatorPBMCs, the other donor will supply responder PBMCs). If desired, thecells are frozen in fetal bovine serum and DMSO after isolation. Frozencells may be thawed overnight in assay media (37° C., 5% CO₂) and thenwashed and resuspended to 3×10⁶ cells/ml of assay media (RPMI; 10% fetalbovine serum, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES, 1%non-essential amino acids, 1% pyruvate). The stimulator PBMCs areprepared by irradiating the cells (about 3000 Rads).

The assay is prepared by plating in triplicate wells a mixture of:

-   -   100:1 of test sample diluted to 1% or to 0.1%,    -   50:1 of irradiated stimulator cells, and    -   50:1 of responder PBMC cells.        100 microliters of cell culture media or 100 microliter of        CD4-IgG is used as the control. The wells are then incubated at        37° C., 5% CO₂ for 4 days. On day 5, each well is pulsed with        tritiated thymidine (1.0 mC/well; Amersham). After 6 hours the        cells are washed 3 times and then the uptake of the label is        evaluated.

In another variant of this assay, PBMCs are isolated from the spleens ofBalb/c mice and C57B6 mice. The. cells are teased from freshly harvestedspleens in assay media (RPMI; 10% fetal bovine serum, 1%penicillin/streptomycin, 1% glutamine, 1% HEPES, 1% non-essential aminoacids, 1% pyruvate) and the PBMCs are isolated by overlaying these cellsover Lympholyte M (Organon Teknika), centrifuging at 2000 rpm for 20minutes, collecting and washing the mononuclear cell layer in assaymedia and resuspending the cells to 1×10⁷ cells/ml of assay media. Theassay is then conducted as described above.

Any decreases below control is considered to be a positive result for aninhibitory compound, with decreases of less than or equal to 80% beingpreferred. However, any value less than control indicates an inhibitoryeffect for the test protein.

The following polypeptide tested positive in this assay: PRO235, PRO245and PRO332.

Example 80 Induction of Endothelial Cell Apoptosis (ELISA) (Assay 109)

The ability of PRO polypeptides to induce apoptosis in endothelial cellswas tested in human venous umbilical vein endothelial cells (HUVEC, CellSystems) using a 96-well format, in 0% serum media supplemented with 100ng/ml VEGF, 0.1% BSA, 1× penn/strep. A positive result in this assayindicates the usefulness of the polypeptide for therapeutically treatingany of a variety of conditions associated with undesired endothelialcell growth including, for example, the inhibition of tumor growth. The96-well plates used were manufactured by Falcon (No. 3072). Coating of96 well plates were prepared by allowing gelatinization to occur for >30minutes with 100 μl of 0.2% gelatin in PBS solution. The gelatin mix wasaspirated thoroughly before plating HUVEC cells at a final concentrationof 2×10⁴ cells/ml in 10% serum containing medium—100 μl volume per well.The cells were grown for 24 hours before adding test samples containingthe PRO polypeptide of interest.

To all wells, 100 μl of 0% serum media (Cell Systems) complemented with100 ng/ml VEGF, 0.1% BSA, 1× penn/strep was added. Test samplescontaining PRO polypeptides were added in triplicate at dilutions of 1%,0.33% and 0.11%. Wells without cells were used as a blank and wells withcells only were used as a negative control. As a positive control, 1:3serial dilutions of 50 of a 3× stock of staurosporine were used. Thecells were incubated for 24 to 35 hours prior to ELISA.

ELISA was used to determine levels of apoptosis preparing solutionsaccording to the Boehringer Manual [Boehringer, Cell Death DetectionELISA plus, Cat No. 1 920 685]. Sample preparations: 96 well plates werespun down at 1 krpm for 10 minutes (200 g); the supernatant was removedby fast inversion, placing the plate upside down on a paper towel toremove residual liquid. To each well, 200 μl of 1× Lysis buffer wasadded and incubation allowed at room temperature for 30 minutes withoutshaking. The plates were spun down for 10 minutes at 1 krpm, and 20 μlof the lysate (cytoplasmic fraction) was transferred into streptavidincoated MTP. 80 μl of immunoreagent mix was added to the 20 μl lystate ineach well. The MTP was covered with adhesive foil and incubated at roomtempearature for 2 hours by placing it on an orbital shaker (200 rpm).After two hours, the supernatant was removed by suction and the wellsrinsed three times with 250 μl of 1× incubation buffer per well (removedby suction). Substrate solution was added (100 μl) into each well andincubated on an orbital shaker at room temperature at 250 rpm untilcolor development was sufficient for a photometric analysis (approx.after 10-20 minutes). A 96 well reader was used to read the plates at405 nm, reference wavelength, 492 nm. The levels obtained for PIN 32(control buffer) was set to 100%. Samples with levels >130% wereconsidered positive for induction of apoptosis.

The following PRO polypeptides tested positive in this assay: PRO235.

Example 81 Human Venous Endothelial Cell Calcium Flux Assay (Assay 68)

This assay is designed to determine whether PRO polypeptides of thepresent invention show the ability to stimulate calcium flux in humanumbilical vein endothelial cells (HUVEC, Cell Systems). Calcium influxis a well documented response upon binding of certain ligands to theirreceptors. A test compound that results in a positive response in thepresent calcium influx assay can be said to bind to a specific receptorand activate a biological signaling pathway in human endothelial cells.This could ultimately lead, for example, to endothelial cell division,inhibition of endothelial cell proliferation, endothelial tubeformation, cell migration, apoptosis, etc.

Human venous umbilical vein endothelial cells (HUVEC, Cell Systems) ingrowth media (50:50 without glycine, 1% glutamine, 10 mM Hepes, 10% FBS,10 ng/ml bFGF), were plated on 96-well microtiter ViewPlates-96 (PackardInstrument Company Part #6005182) microtiter plates at a cell density of2×10⁴ cells/well. The day after plating, the cells were washed threetimes with buffer (HBSS plus 10 MM Hepes), leaving 100 μl/well. Then 100μl/well of 8 μM Fluo-3 (2×) was added. The cells were incubated for 1.5hours at 37° C./5% CO₂. After incubation, the cells were then washed 3×with buffer (described above) leaving 100 μl/well. Test samples of thePRO polypeptides were prepared on different 96-well plates at 5×concentration in buffer. The positive control corresponded to 50 μMionomycin (5×); the negative control corresponded to Protein 32. Cellplate and sample plates were run on a FLIPR (Molecular Devices) machine.The FLIPR machine added 25 μl of test sample to the cells, and readingswere taken every second for one minute, then every 3 seconds for thenext three minutes.

The fluorescence change from baseline to the maximum rise of the curve(Δ change) was calculated, and replicates averaged. The rate offluorescence increase was monitored, and only those samples which had aΔ change greater than 1000 and a rise within 60 seconds, were consideredpositive.

The following PRO polypeptides tested positive in the present assay:PRO245.

Example 82 Fibroblast (BHK-21) Proliferation (Assay 98)

This assay shows that certain PRO polypeptides of the invention act toinduce proliferation of mammalian fibroblast cells in culture and,therefore, function as useful growth factors in mammalian systems. Theassay is performed as follows. BHK-21 fibroblast cells plated instandard growth medium at 2500 cells/well in a total volume of 100 μl.The PRO polypeptide, P-FGF (positive control) or nothing (negativecontrol) are then added to the wells in the presence of 1 μg/ml ofheparin for a total final volume of 200 μl. The cells are then incubatedat 37° C. for 6 to 7 days. After incubation, the media is removed, thecells are washed with PBS and then an acid phosphatase substratereaction mixture (100 μl/well) is added. The cells are then incubated at37° C. for 2 hours. 10 μl per well of 1N NaOH is then added to stop theacid phosphatase reaction. The plates are then read at OD 405 nm. Apositive in the assay is acid phosphatase activity which is at least 50%above the negative control.

The following PRO polypeptide tested positive in this assay: PRO258.

Example 83 Inhibition of Heart Adult Hypertrophy (Assay 42)

This assay is designed to measure the inhibition of heart adulthypertrophy. PRO polypeptides testing positive in this assay may finduse in the therapeutic treatment of cardiac disorders associated withcardiac hypertrophy.

Ventricular myocytes are freshly isolated from adult (250 g) HarlanSprague Dawley rats and the cells are plated at 2000/well in 180 μlvolume. On day two, test samples (20 μl) containing the test PROpolypeptide are added. On day five, the cells are fixed and thenstained. An increase in ANP message can also be measured by PCR fromcells after a few hours. Results are based on a visual score of cellsize: 0=no inhibition, −1=small inhibition, −2=large inhibition. A scoreof less than 0 is considered positive. Activity reference corresponds tophenylephrin (PE) at 0.1 mM, as a positive control. Assay mediaincluded: M199 (modified)-glutamine free, NaHCO₃, phenol red,supplemented with 100 nM insulin, 0.2% BSA, 5 mM cretine, 2 mML-carnitine, 5 mM taurine, 100 U/ml penicillin G, 100 μg/ml streptomycin(CCT medium). Only inner 60 wells are used in 96 well plates. Of these,6 wells are reserved for negative and positive (PE) controls.

The following PRO polypeptides provided a score of less than 0 in theabove assay: PRO269.

Example 84 Induction of c-fos in Endothelial Cells (Assay 34)

This assay is designed to determine whether PRO polypeptides show theability to induce c-fos in endothelial cells. PRO polypeptides testingpositive in this assay would be expected to be useful for thetherapeutic treatment of conditions or disorders where angiogenesiswould be beneficial including, for example, wound healing, and the like(as would agonists of these PRO polypeptides). Antagonists of the PROpolypeptides testing positive in this assay would be expected to beuseful for the therapeutic treatment of cancerous tumors.

Human venous umbilical vein endothelial cells (HUVEC, Cell Systems) ingrowth media (50% Ham's F12 w/o GHT: low glucose, and 50% DMEM withoutglycine: with NaHCO3, 1% glutamine, 10 mM HEPES, 10% FBS, 10 ng/ml bFGF)were plated on 96-well microtiter plates at a cell density of 1×10⁴cells/well. The day after plating, the cells were starved by removingthe growth media and treating the cells with 100 μl/well test samplesand controls (positive control=growth media; negative control=Protein 32buffer=10 mM HEPES, 140 mM NaCl, 4% (w/v) mannitol, pH 6.8). The cellswere incubated for 30 minutes at 37° C., in 5% CO₂. The samples wereremoved, and the first part of the bDNA kit protocol (ChironDiagnostics, cat. #6005-037) was followed, where each capitalizedreagent/buffer listed below was available from the kit.

Briefly, the amounts of the TM Lysis Buffer and Probes needed for thetests were calculated based on information provided by the manufacturer.The appropriate amounts of thawed Probes were added to the TM LysisBuffer. The Capture Hybridization Buffer was warmed to room temperature.The bDNA strips were set up in the metal strip holders, and 100 μl ofCapture Hybridization Buffer was added to each b-DNA well needed,followed by incubation for at least 30 minutes. The test plates with thecells were removed from the incubator, and the media was gently removedusing the vacuum manifold. 100 μl of Lysis Hybridization Buffer withProbes were quickly pipetted into each well of the microtiter plates.The plates were then incubated at 55° C. for 15 minutes. Upon removalfrom the incubator, the plates were placed on the vortex mixer with themicrotiter adapter head and vortexed on the #2 setting for one minute.80 μl of the lysate was removed and added to the bDNA wells containingthe Capture Hybridization Buffer, and pipetted up and down to mix. Theplates were incubated at 53° C. for at least 16 hours.

On the next day, the second part of the bDNA kit protocol was followed.Specifically, the plates were removed from the incubator and placed onthe bench to cool for 10 minutes. The volumes of additions needed werecalculated based upon information provided by the manufacturer. AnAmplifier Working Solution was prepared by making a 1:100 dilution ofthe Amplifier Concentrate (20 fm/μl) in AL Hybridization Buffer. Thehybridization mixture was removed from the plates and washed twice withWash A. 50 μl of Amplifier Working Solution was added to each well andthe wells were incubated at 53° C. for 30 minutes. The plates were thenremoved from the incubator and allowed to cool for 10 minutes. The LabelProbe Working Solution was prepared by making a 1:100 dilution of LabelConcentrate (40 pmoles/μl) in AL Hybridization Buffer. After the10-minute cool-down period, the amplifier hybridization mixture wasremoved and the plates were washed twice with Wash A. 50 μl of LabelProbe Working Solution was added to each well and the wells wereincubated at 53° C. for 15 minutes. After cooling for 10 minutes, theSubstrate was warmed to room temperature. Upon addition of 3 μl ofSubstrate Enhancer to each ml of Substrate needed for the assay, theplates were allowed to cool for 10 minutes, the label hybridizationmixture was removed, and the plates were washed twice with Wash A andthree times with Wash D. 50 μl of the Substrate Solution with Enhancerwas added to each well. The plates were incubated for 30 minutes at 37°C. and RLU was read in an appropriate luminometer.

The replicates were averaged and the coefficient of variation wasdetermined. The measure of activity of the fold increase over thenegative control (Protein 32/HEPES buffer described above) value wasindicated by chemiluminescence units (RLU). The results are consideredpositive if the PRO polypeptide exhibits at least a two-fold value overthe negative buffer control. Negative control=1.00 RLU at 1.00%dilution. Positive control=8.39 RLU at 1.00% dilution.

The following PRO polypeptides tested positive in this assay: PRO287.

Example 85 Guinea Pig Vascular Leak (Assays 32 and 51)

This assay is designed to determine whether PRO polypeptides of thepresent invention show the ability to induce vascular permeability.Polypeptides testing positive in this assay are expected to be usefulfor the therapeutic treatment of conditions which would benefit fromenhanced vascular permeability including, for example, conditions whichmay benefit from enhanced local immune system cell infiltration.

Hairless guinea pigs weighing 350 grams or more were anesthetized withKetamine (75-80 mg/kg) and 5 mg/kg Xylazine intramuscularly. Testsamples containing the PRO polypeptide or a physiological buffer withoutthe test polypeptide are injected into skin on the back of the testanimals with 100 μl per injection site intradermally. There wereapproximately 16-24 injection sites per animal. One ml of Evans blue dye(1% in PBS) is then injected intracardially. Skin vascular permeabilityresponses to the compounds (i.e., blemishes at the injection sites ofinjection) are visually scored by measuring the diameter (in mm) ofblue-colored leaks from the site of injection at 1 and 6 hours postadministration of the test materials. The mm diameter of blueness at thesite of injection is observed and recorded as well as the severity ofthe vascular leakage. Blemishes of at least 5 mm in diameter areconsidered positive for the assay when testing purified proteins, beingindicative of the ability to induce vascular leakage or permeability. Aresponse greater than 7 mm diameter is considered positive forconditioned media samples. Human VEGF at 0.1 μg/100 μl is used as apositive control, inducing a response of 15-23 mm diameter.

The following PRO polypeptides tested positive in this assay: PRO302 andPRO533.

Example 86 Detection of Endothelial Cell Apoptosis (FACS) (Assay 96)

The ability of PRO polypeptides of the present invention to induceapoptosis in endothelial cells was tested in human venous umbilical veinendothelial cells (HUVEC, Cell Systems) in gelatinized T175 flasks usingHUVEC cells below passage 10. PRO polypeptides testing positive in thisassay are expected to be useful for therapeutically treating conditionswhere apoptosis of endothelial cells would be beneficial including, forexample, the therapeutic treatment of tumors.

On day one, the cells were split [420,000 cells per gelatinized 6 cmdishes−(11×10³ cells/cm² Falcon, Primaria)] and grown in mediacontaining serum (CS-C, Cell System) overnight or for 16 hours to 24hours.

On day 2, the cells were washed 1× with 5 ml PBS; 3 ml of 0% serummedium was added with VEGF (100 ng/ml); and 30 μl of the PRO testcompound (final dilution 1%) or 0% serum medium (negative control) wasadded. The mixtures were incubated for 48 hours before harvesting.

The cells were then harvested for FACS analysis. The medium wasaspirated and the cells washed once with PBS. 5 ml of 1× trypsin wasadded to the cells in a T-175 flask, and the cells were allowed to standuntil they were released from the plate (about 5-10 minutes).Trypsinization was stopped by adding 5 ml of growth media. The cellswere spun at 1000 rpm for 5 minutes at 4° C. The media was aspirated andthe cells were resuspended in 10 ml of 10% serum complemented medium(Cell Systems), 5 μl of Annexin-FITC (BioVison) added and chilled tubeswere submitted for FACS. A positive result was determined to be enhancedapoptosis in the PRO polypeptide treated samples as compared to thenegative control.

The following PRO polypeptides tested positive in this assay: PRO331.

Example 87 Induction of c-fos in Cortical Neurons (Assay 83)

This assay is designed to determine whether PRO polypeptides show theability to induce c-fos in cortical neurons. PRO polypeptides testingpositive in this assay would be expected to be useful for thetherapeutic treatment of nervous system disorders and injuries whereneuronal proliferation would be beneficial.

Cortical neurons are dissociated and plated in growth medium at 10,000cells per well in 96 well plates. After aproximately 2 cellulardivisions, the cells are treated for 30 minutes with the PRO polypeptideor nothing (negative control). The cells are then fixed for 5 minuteswith cold methanol and stained with an antibody directed againstphosphorylated CREB. mRNA levels are then calculated usingchemiluminescence. A positive in the assay is any factor that results inat least a 2-fold increase in c-fos message as compared to the negativecontrols.

The following PRO polypeptides tested positive in this assay: PRO229 andPRO269.

Example 88 Stimulation of Endothelial Tube Formation (Assay 85)

This assay is designed to determine whether PRO polypeptides show theability to promote endothelial vacuole and lumen formation in theabsence of exogenous growth factors. PRO polypeptides testing positivein this assay would be expected to be useful for the therapeutictreatment of disorders where endothelial vacuole and/or lumen formationwould be beneficial including, for example, where the stimulation ofpinocytosis, ion pumping, vascular permeability and/or junctionalformation would be beneficial.

HUVEC cells (passage <8 from primary) are mixed with type I rat tailcollagen (final concentration 2.6 mg/ml) at a density of 6×10⁵ cells perml and plated at 50 μl per well of M199 culture media supplemented with1% FBS and 1 μM 6-FAM-FITC dye to stain the vacuoles while they areforming and in the presence of the PRO polypeptide. The cells are thenincubated at 37° C./5% CO₂ for 48 hours, fixed with 3.7% formalin atroom temperature for 10 minutes, washed 5 times with M199 medium andthen stained with Rh-Phalloidin at 4° C. overnight followed by nuclearstaining with 4 μM DAPI. A positive result in the assay is when vacuolesare present in greater than 50% of the cells.

The following PRO polypeptides tested positive in this assay: PRO230.

Example 89 Detection of Polypeptides hat Affect Glucose and/or FFAUptake in Skeletal Muscle (Assay 106)

This assay is designed to determine whether PRO polypeptides show theability to affect glucose or FFA uptake by skeletal muscle cells. PROpolypeptides testing positive in this assay would be expected to beuseful for the therapeutic treatment of disorders where either thestimulation or inhibition of glucose uptake by skeletal muscle would bebeneficial including, for example, diabetes or hyper- orhypo-insulinemia.

In a 96 well format, PRO polypeptides to be assayed are added to primaryrat differentiated skeletal muscle, and allowed to incubate overnight.Then fresh media with the PRO polypeptide and +/−insulin are added tothe wells. The sample media is then monitored to determine glucose andFFA uptake by the skeletal muscle cells. The insulin will stimulateglucose and FFA uptake by the skeletal muscle, and insulin in mediawithout the PRO polypeptide is used as a positive control, and a limitfor scoring. As the PRO polypeptide being tested may either stimulate orinhibit glucose and FFA uptake, results are scored as positive in theassay if greater than 1.5 times or less than 0.5 times the insulincontrol.

The following PRO polypeptides tested positive as either stimulators orinhibitors of glucose and/or FFA uptake in this assay: PRO187, PRO211,PRO221, PRO222, PRO224, PRO230, PRO239, PRO231, PRO245, PRO247, PRO258,PRO269, PRO328 and PRO533.

Example 90 Rod Photoreceptor Cell Survival Assay (Assay 46)

This assay shows that certain polypeptides of the invention act toenhance the survival/proliferation of rod photoreceptor cells and,therefore, are useful for the therapeutic treatment of retinal disordersor injuries including, for example, treating sight loss in mammals dueto retinitis pigmentosum, AMD, etc.

Sprague Dawley rat pups (postnatal day 7, mixed population: glia andnetinal neural cell types) are killed by decapitation following CO₂anesthesia and the eyes removed under sterile conditions. The neuralretina is dissected away from the pigment epithelium and other oculartissue and then dissociated into a single cell suspension using 0.25%trypsin in Ca²⁺, Mg²⁺-free PBS. The retinas are incubated at 37° C. inthis solution for 7-10 minutes after which the trypsin is inactivated byadding 1 ml soybean trypsin inhibitor. The cells are plated at a densityof approximately 10,000 cells/ml into 96 well plates in DMEM/F12supplemented with N₂. Cells for all experiments are grown at 37° C. in awater saturated atmosphere of 5% CO₂. After 7-10 days in culture, thecells are stained using calcein AM or CellTracker Green CMFDA and thenfixed using 4% paraformaldehyde. Rho 4D2 (ascities or IgG 1:100)monoclonal antibody directed towards the visual pigment rhodopsin isused to detect rod photoreceptor cells by indirect immunofluorescence.The results are calculated as % survival: total number ofcalcein—rhodopsin positive cells at 7-10 days in culture, divided by thetotal number of rhodopsin positive cells at time 7-10 days in culture.The total cells (fluorescent) are quantified at 20× objectivemagnification using a CCD camera and NIH image software for MacIntosh.Fields in the well are chosen at random.

The following polypeptides tested positive in this assay: PRO245.

Example 91 In vitro Antitumor Assay (Assay 161)

The antiproliferative activity of various PRO polypeptides wasdetermined in the investigational, disease-oriented in vitro anti-cancerdrug discovery assay of the National Cancer Institute (NCI), using asulforhodamine B (SRB) dye binding assay essentially as described bySkehan et al., J. Natl. Cancer Inst. 82:1107-1112 (1990). The 60 tumorcell lines employed in this study (“the NCI panel”), as well asconditions for their maintenance and culture in vitro have beendescribed by Monks et al., J. Natl. Cancer Inst. 83:757-766 (1991). Thepurpose of this screen is to initially evaluate the cytotoxic and/orcytostatic activity of the test compounds against different types oftumors (Monks et al., supra; Boyd, Cancer: Princ. Pract. Oncol. Update3(10):1-12 [1989]).

Cells from approximately 60 human tumor cell lines were harvested withtrypsin/EDTA (Gibco), washed once, resuspended in IMEM and theirviability was determined. The cell suspensions were added by pipet (100μL volume) into separate 9-well microtiter plates. The cell density forthe 6-day incubation was less than for the 2-day incubation to preventovergrowth. Inoculates were allowed a preincubation period of 24 hoursat 37° C. for stabilization. Dilutions at twice the intended testconcentration were added at time zero in 100 μL aliquots to themicrotiter plate wells (1:2 dilution). Test compounds were evaluated atfive half-log dilutions (1000 to 100,000-fold). Incubations took placefor two days and six days in a 5% CO₂ atmosphere and 100% humidity.

After incubation, the medium was removed and the cells were fixed in 0.1ml of 10% trichloroacetic acid at 40° C. The plates were rinsed fivetimes with deionized water, dried, stained for 30 minutes with 0.1 ml of0.4% sulforhodamine B dye (Sigma) dissolved in 1% acetic acid, rinsedfour times with 1% acetic acid to remove unbound dye, dried, and thestain was extracted for five minutes with 0.1 ml of 10 mM Tris base[tris(hydroxymethyl)aminomethane], pH 10.5. The absorbance (OD) ofsulforhodamine B at 492 nm was measured using a computer-interfaced,96-well microtiter plate reader.

A test sample is considered positive if it shows at least 50% growthinhibitory effect at one or more concentrations. PRO polypeptidestesting positive in this assay are shown in Table 7, where theabbreviations are as follows:

-   NSCL:=non-small cell lung carcinoma-   CNS=central nervous system

TABLE 7 Test compound Tumor Cell Line Type Cell Line Designation PRO211NSCL HOP62 PRO211 Leukemia RPMI-8226 PRO211 Leukemia HL-60 (TB) PRO211NSCL NCI-H522 PRO211 CNS SF-539 PRO211 Melanoma LOX IMVI PRO211 BreastMDA-MB-435 PRO211 Leukemia MOLT-4 PRO211 CNS U251 PRO211 Breast MCF7PRO211 Leukemia HT-60 (TB) PRO211 Leukemia MOLT-4 PRO211 NSCL EKVXPRO211 NSCL NCI-H23 PRO211 NSCL NCI-H322M PRO211 NSCL NCI-H460 PRO211Colon HCT-116 PRO211 Colon HT29 PRO211 CNS SF-268 PRO211 CNS SF-295PRO211 CNS SNB-19 PRO211 CNS U251 PRO211 Melanoma LOX IMVI PRO211Melanoma SK-MEL-5 PRO211 Melanoma UACC-257 PRO211 Melanoma UACC-62PRO211 Ovarian OVCAR-8 PRO211 Renal RXF 393 PRO211 Breast MCF7 PRO211Breast NCI/ADR-REHS 578T PRO211 Breast T-47D PRO211 Leukemia HL-60 (TB)PRO211 Leukemia SR PRO211 NSCL NCI-H23 PRO211 Colon HCT-116 PRO211Melanoma LOX-IMVI PRO211 Melanoma SK-MEL-5 PRO211 Breast T-47D PRO228Leukemia MOLT-4 PRO228 NSCL EKVX PRO228 Colon KM12 PRO228 MelanomaUACC-62 PRO228 Ovarian OVCAR-3 PRO228 Renal TK10 PRO228 Renal SN12CPRO228 Breast MCF7 PRO228 Leukemia CCRF-CEM PRO228 Leukemia HL-60 (TB)PRO228 Colon COLO 205 PRO228 Colon HCT-15 PRO228 Colon KM12 PRO228 CNSSF-268 PRO228 CNS SNB-75 PRO228 Melanoma LOX-IMVI PRO228 MelanomaSK-MEL2 PRO228 Melanoma UACC-257 PRO228 Ovarian IGROV1 PRO228 OvarianOVCAR-4 PRO228 Ovarian OVCAR-5 PRO228 Ovarian OVCAR-8 PRO228 Renal 786-0PRO228 Renal CAKI-1 PRO228 Renal RXF 393 PRO228 Renal TK-10 PRO228 RenalUO-31 PRO228 Prostate PC-3 PRO228 Prostate DU-145 PRO228 Breast MCF7PRO228 Breast NCI/ADR-REHS 578T PRO228 Breast MDA-MB-435MDA-N PRO228Breast T-47D PRO219 Leukemia SR PRO219 NSCL NCI-H5222 PRO219 Breast MCF7PRO219 Leukemia K-562; RPMI-8226 PRO219 NSCL HOP-62; NCI-H322M PRO219NSCL NCI -H460 PRO219 Colon HT29; KM12; HCT-116 PRO219 CNS SF-539; U251PRO219 Prostate DU-145 PRO219 Breast MDA-N PRO219 Ovarian IGROV1 PRO219NSCL NCI-H226 PRO219 Leukemia MOLT-4 PRO219 NSCL A549/ATCC; EKVX;NCI-H23 PRO219 Colon HCC-2998 PRO219 CNS SF-295; SNB-19 PRO219 MelanomaSK-MEL-2; SK-MEL-5 PRO219 Melanoma UACC-257; UACC-62 PRO219 OvarianOCAR-4; SK-OV-3 PRO219 Renal 786-0; ACHN; CAKI-1; SN12C PRO219 RenalTK-10; UO-31 PRO219 Breast NCI/ADR-RES; BT-549; T-47D PRO219 BreastMDA-MB-435 PRO221 Leukemia CCRF-CEM PRO221 Leukemia MOLT-4 PRO221 NSCLHOP-62 PRO221 Breast MDA-N PRO221 Leukemia RPMI-8226; SR PRO221 NSCLNCI-H460 PRO221 Colon HCC-2998 PRO221 Ovarian IGROV1 PRO221 Renal TK-10PRO221 Breast MCF7 PRO221 Leukemia K-562 PRO221 Breast MDA-MB-435 PRO224Ovarian OVCAR-4 PRO224 Renal RXF 393 PRO224 Prostate DU-145 PRO224 NSCLHOP-62; NCI-H322M PRO224 Melanoma LOX IMVI PRO224 Ovarian OVCAR-8 PRO224Leukemia SR PRO224 NSCL NCI-H460 PRO224 CNS SF-295 PRO224 LeukemiaRPMI-8226 PRO224 Breast BT-549 PRO224 Leukemia CCRF-CEM; LH-60 (TB)PRO224 Colon HCT-116 PRO224 Breast MDA-MB-435 PRO224 Leukemia HL-60 (TB)PRO224 Colon HCC-2998 PRO224 Prostate PC-3 PRO224 CNS U251 PRO224 ColonHCT-15 PRO224 CNS SF-539 PRO224 Renal ACHN PRO328 Leukemia RPMI-8226PRO328 NSCL A549/ATCC; EKVX; HOP-62 PRO328 NSCL NCI-H23; NCI-H322MPRO328 Colon HCT-15; KM12 PRO328 CNS SF-295; SF-539; SNB-19; U251 PRO328Melanoma M14; UACC-257; UCAA-62 PRO328 Renal 786-0; ACHN PRO328 BreastMCF7 PRO328 Leukemia SR PRO328 Colon NCI-H23 PRO328 Melanoma SK-MEL-5PRO328 Prostate DU-145 PRO328 Melanoma LOX IMVI PRO328 Breast MDA-MB-435PRO328 Ovarian OVCAR-3 PRO328 Breast T-47D PRO301 NSCL NCI-H322M PRO301Leukemia MOLT-4; SR PRO301 NSCL A549/ATCC; EKVX; PRO301 NSCL NCI-H23;NCI-460; NCI-H226 PRO301 Colon COLO 205; HCC-2998; PRO301 Colon HCT-15;KM12; HT29; PRO301 Colon HCT-116 PRO301 CNS SF-268; SF-295; SNB-19PRO301 Melanoma MALME-3M; SK-MEL-2; PRO301 Melanoma SK-MEL-5;UACC-257PRO301 Melanoma UACC-62 PRO301 Ovarian IGROV1; OVCAR-4 PRO301 OvarianOVCAR-5 PRO301 Ovarian OVCAR-8; SK0OV-3 PRO301 Renal ACHN;CAKI-1; TK-10;UO-31 PRO301 Prostate PC-3; DU-145 PRO301 Breast NCI/ADR-RES; HS 578TPRO301 Breast MDA-MB-435;MDA-N; T-47D PRO301 Melanoma M14 PRO301Leukemia CCRF-CEM;HL-60(TB); K-562 PRO301 Leukemia RPMI-8226 PRO301Melanoma LOX IMVI PRO301 Renal 786-0; SN12C PRO301 Breast MCF7;MDA-MB-231/ATCC PRO301 Breast BT-549 PRO301 NSCL HOP-62 PRO301 CNSSF-539 PRO301 Ovarian OVCAR-3 PRO326 NSCL NCI-H322M PRO326 CNS SF295PRO326 CNS ST539 PRO326 CNS U251

The results of these assays demonstrate that the positive testing PROpolypeptides are useful for inhibiting neoplastic growth in a number ofdifferent tumor cell types and may be used therapeutically therefor.Antibodies against these PRO polypeptides are useful for affinitypurification of these useful polypeptides. Nucleic acids encoding thesePRO polypeptides are useful for the recombinant preparation of thesepolypeptides.

Example 92 Gene Amplification

This example shows that certain PRO polypeptide-encoding genes areamplified in the genome of certain human lung, colon and/or breastcancers and/or cell lines. Amplification is associated withoverexpression of the gene product, indicating that the polypeptides areuseful targets for therapeutic intervention in certain cancers such ascolon, lung, breast and other cancers and diagnostic determination ofthe presence of those cancers. Therapeutic agents may take the form ofantagonists of the PRO polypeptide, for example, murine-human chimeric,humanized or human antibodies against a PRO polypeptide.

The starting material for the screen was genomic DNA isolated from avariety cancers. The DNA is quantitated precisely, e.g.,fluorometrically. As a negative control, DNA was isolated from the cellsof ten normal healthy individuals which was pooled and used as assaycontrols for the gene copy in healthy individuals (not shown). The 5′nuclease assay (for example, TaqMan™) and real-time quantitative PCR(for example, ABI Prizm 7700 Sequence Detection Systems (Perkin Elmer,Applied Biosystems Division, Foster City, Calif.)), were used to findgenes potentially amplified in certain cancers. The results were used todetermine whether the DNA encoding the PRO polypeptide isover-represented in any of the primary lung or colon cancers or cancercell lines or breast cancer cell lines that were screened. The primarylung cancers were obtained from individuals with tumors of the type andstage as indicated in Table 8. An explanation of the abbreviations usedfor the designation of the primary tumors listed in Table 8 and theprimary tumors and cell lines referred to throughout this example aregiven below.

The results of the TaqMan™ are reported in delta (Δ) Ct units. One unitcorresponds to 1 PCR cycle or approximately a 2-fold amplificationrelative to normal, two units corresponds to 4-fold, 3 units to 8-foldamplification and so on. Quantitation was obtained using primers and aTaqMan™ fluorescent probe derived from the PRO polypeptide-encodinggene. Regions of the PRO polypeptide-encoding gene which are most likelyto contain unique nucleic acid sequences and which are least likely tohave spliced out introns are preferred for the primer and probederivation, e.g., 3′-untranslated regions. The sequences for the primersand probes (forward, reverse and probe) used for the PRO polypeptidegene amplification analysis were as follows:

PRO187 (DNA27864-1155) 27864.tm.p: 5-GCAGATTTTGAGGACAGCCACCTCCA-3′ (SEQID NO:381) 27864.tm.f: 5′-GGCCTTGCAGACAACCGT-3′ (SEQ ID NO:382)27864.tm.r: 5′-CAGACTGAGGGAGATCCGAGA-3′ (SEQ ID NO:383) 27864.tm.p2:5′-CAGCTGCCCTTCCCCAACCA-3′ (SEQ ID NO:384) 27864.tm.f2:5′-CATCAAGCGCCTCTACCA-3′ (SEQ ID NO:385) 27864.tm.r2:5′-CACAAACTCGAACTGCTTCTG-3′ (SEQ ID NO:386) PRO214 (DNA32286-1191):32286.3utr-5: 5′-GGGCCATCACAGCTCCCT-3′ (SEQ ID NO:387) 32286.3utr-3b:5′-GGGATGTGGTGAACACAGAACA-3′ (SEQ ID NO:388) 32286.3utr-probe:5′-TGCCAGCTGCATGCTGCCAGTT-3′ (SEQ ID NO:389) PRO211 (DNA32292-1131):32292.3utr-5: 5′-CAGAAGGATGTCCCGTGGAA-3′ (SEQ ID NO:390) 32292.3utr-3:5′-GCCGCTGTCCACTGCAG-3′ (SEQ ID NO:391) 32292.3utr-probe.rc:5′-GACGGCATCCTCAGGGCCACA-3′ (SEQ ID NO:392) PRO230 (DNA33223-1136):33223.tm.p3: 5′-ATGTCCTCCATGCCCACGCG-3′ (SEQ ID NO:393) 33223.tm.f3:5′-GAGTGCGACATCGAGAGCTT-3′ (SEQ ID NO:394) 33223.tm.r3:5′-CCGCAGCCTCAGTGATGA-3′ (SEQ ID NO:395) 33223.3utr-5:5′-GAAGAGCACAGCTGCAGATCC-3′ (SEQ ID NO:396) 33223.3utr-3:5′-GAGGTGTCCTGGCTTTGGTAGT-3′ (SEQ ID NO:397) 33223.3utr-probe:5′-CCTCTGGCGCCCCCACTCAA-3′ (SEQ ID NO:398) PRO317 (DNA33461-1199):33461.tm.f: 5′-CCAGGAGAGCTGGCGATG-3′ (SEQ ID NO:399) 33461.tm.r:5′-GCAAATTCAGGGCTCACTAGAGA-3′ (SEQ ID NO:400) 33461.tm.p:5′-CACAGAGCATTTGTCCATCAGCAGTTCAG-3′ (SEQ ID NO:401) PRO246(DNA35639-1172): 35639.3utr-5: 5′-GGCAGAGACTTCCAGTCACTGA-3′ (SEQ IDNO:402) 35639.3utr-3: 5′-GCCAAGGGTGGTGTTAGATAGG-3′ (SEQ ID NO:403)35639.3utr-probe: 5′-CAGGCCCCCTTGATCTGTACCCCA-3′ (SEQ ID NO:404) PRO533(DNA49435-1219): 49435.tm.f: 5′-GGGACGTGCTTCTACAAGAACAG-3′ (SEQ IDNO:405) 49435.tm.r: 5′-CAGGCTTACAATGTTATGATCAGACA-3′ (SEQ ID NO:406)49435.tm.p: 5′-TATTCAGAGTTTTCCATTGGCAGTGCCAGTT-3′ (SEQ ID NO:407) PRO343(DNA43318-1217): 43318.tm.f1 5′-TCTACATCAGCCTCTCTGCGC-3′ (SEQ ID NO:408)43318.tm.p1 5′-CGATCTTCTCCACCCAGGAGCGG-3′ (SEQ ID NO:409) 433 18.tm.r15′-GGAGCTGCACCCCTTGC-3′ (SEQ ID NQ:237) PRO232 (DNA34435-1140):34435.3utr-5: 5′-GCCAGGCCTCACATTCGT-3′ (SEQ ID NO:410)DNA34435.3utr-probe: 5′-CTCCCTGAATGGCAGCCTGAGCA-3′ (SEQ ID NO:411)DNA34435.3utr-3: 5′-AGGTGTTTATTAAGGGCCTACGCT-3′ (SEQ ID NO:412) PRO269(DNA38260-1180): 38260.tm.f: 5′-CAGAGCAGAGGGTGCCTTG-3′ (SEQ ID NO:41338260.tm.p: 5′-TGGCGGAGTCCCCTCTTGGCT-3′ (SEQ ID NO:414) 38260.tm.r:5′-CCCTGTTTCCCTATGCATCACT-3′ (SEQ ID NO:415) PRO304 (DNA39520-1217):39520.tm.f: 5′-TCAACCCCTGACCCTTTCCTA-3′ (SEQ ID NO:416) 3952O.tm.p:5′-GGCAGGGGACAAGCCATCTCTCCT-3′ (SEQ ID NO:417) 39520.tm.r:5′-GGGACTGAACTGCCAGCTTC-3′ (SEQ ID NO:418) PRO339 (DNA43466-1225):43466.tm.f1: 5′-GGGCCCTAACCTCATTACCTTT-3′ (SEQ ID NO:419) 43466.tm.p1:5′-TGTCTGCCTCAGCCCCAGGAAGG-3′ (SEQ ID NO:420) 43466.tm.r1:5′-TCTGTCCACCATCTTGCCTTG -3′ (SEQ ID NO:421)

The 5′ nuclease assay reaction is a fluorescent PCR-based techniquewhich makes use of the 5′ exonuclease activity of Taq DNA polymeraseenzyme to monitor amplification in real time. Two oligonucleotideprimers (forward [.f] and reverse [.r]) are used to generate an amplicontypical of a PCR reaction. A third oligonucleotide, or probe (.p), isdesigned to detect nucleotide sequence located between the two PCRprimers. The probe is non-extendible by Taq DNA polymerase enzyme, andis labeled with a reporter fluorescent dye and a quencher fluorescentdye. Any laser-induced emission from the reporter dye is quenched by thequenching dye when the two dyes are located close together as they areon the probe. During the amplification reaction, the Taq DNA polymeraseenzyme cleaves the probe in a template-dependent manner. The resultantprobe fragments disassociate in solution, and signal from the releasedreporter dye is free from the quenching effect of the secondfluorophore. One molecule of reporter dye is liberated for each newmolecule synthesized, and detection of the unquenched reporter dyeprovides the basis for quantitative interpretation of the data.

The 5′ nuclease procedure is run on a real-time quantitative PCR devicesuch as the ABI Prism 7700TM Sequence Detection. The system consists ofa thermocycler, laser, charge-coupled device (CCD) camera and computer.The system amplifies samples in a 96-well format on a thermocycler.During amplification, laser-induced fluorescent signal is collected inreal-time through fiber optics cables for all 96 wells, and detected atthe CCD. The system includes software for running the instrument and foranalyzing the data.

5′ Nuclease assay data are initially expressed as Ct, or the thresholdcycle. This is defined as the cycle at which the reporter signalaccumulates above the background level of fluorescence. The ΔCt valuesare used as quantitative measurement of the relative number of startingcopies of a particular target sequence in a nucleic acid sample whencomparing cancer DNA results to normal human DNA results.

Table 8 describes the stage, T stage and N stage of various primarytumors which were used to screen the PRO polypeptide compounds of theinvention.

TABLE 8 Primary Lung and Colon Tumor Profiles Primary Tumor Stage StageOther Stage Dukes Stage T Stage N Stage Human lung tumor AdenoCa(SRCC724) [LT1] IIA T1 N1 Human lung tumor SqCCa (SRCC725) [LT1a] IIB T3N0 Human lung tumor AdenoCa (SRCC726) [LT2] IB T2 N0 Human lung tumorAdenoCa (SRCC727) [LT3] IIIA T1 N2 Human lung tumor AdenoCa (SRCC728)[LT4] IB T2 N0 Human lung tumor SqCCa (SRCC729) [LT6] IB T2 N0 Humanlung tumor Aden/SqCCa (SRCC730) [LT7] IA T1 N0 Human lung tumor AdenoCa(SRCC731) [LT9] IB T2 N0 Human lung tumor SqCCa (SRCC732) [LT10] IIB T2N1 Human lung tumor SqCCa (SRCC733) [LT11] IIA T1 N1 Human lung tumorAdenoCa (SRCC734) [LT12] IV T2 N0 Human lung tumor AdenoSqCCa (5RCC735)[LT13] IB T2 N0 Human lung tumor SqCCa (SRCC736) [LT15] IB T2 N0 Humanlung tumor SqCCa (SRCC737) [LT16] IB T2 N0 Human lung tumor SqCCa(SRCC738) [LT17] IIB T2 N1 Human lung tumor SqCCa (SRCC739) [LT18] IB T2N0 Human lung tumor SqCCa (SRCC740) [LT19] IB T2 N0 Human lung tumorLCCa (SRCC74l) [LT21] IIB T3 N1 Human lung AdenoCa (SRCC811) [LT22] 1AT1 N0 Human colon AdenoCa (SRCC742) [CT2] M1 D pT4 N0 Human colonAdenoCa (SRCC743) [CT3] B pT3 N0 Human colon AdenoCa (SRCC744) [CT8] BT3 N0 Human colon AdenoCa (SRCC745) [CT10] A pT2 N0 Human colon AdenoCa(SRCC746) [CT12] MO, R1 B T3 N0 Human colon AdenoCa (SRCC747) [CT14]pMO, RO B pT3 pN0 Human colon AdenoCa (SRCC748) [CT15] M1, R2 D T4 N2Human colon AdenoCa (SRCC749) [CT16] pMO B pT3 pN0 Human colon AdenoCa(SRCC750) [CT17] C1 pT3 pN1 Human colon AdenoCa (SRCC751) [CT1] MO, R1 BpT3 N0 Human colon AdenoCa (SRCC752) [CT4] B pT3 M0 Human colon AdenoCa(SRCC753) [CT5] G2 C1 p13 pN0 Human colon AdenoCa (SRCC754) [CT6] pMO,RO B pT3 pN0 Human colon AdenoCa (SRCC755) [CT7] G1 A pT2 pN0 Humancolon AdenoCa (SRCC756) [CT9] G3 D pT4 pN2 Human colon AdenoCa (SRCC757)[CT11] B T3 N0 Human colon AdenoCa (SRCC758) [CT18] MO, RO B pT3 pN0DNA Preparation

DNA was prepared from cultured cell lines, primary tumors, normal humanblood. The isolation was performed using purification kit, buffer setand protease and all from Quiagen, according to the manufacturer'sinstructions and the description below.

Cell Culture Lysis

Cells were washed and trypsinized at a concentration of 7.5×10⁸ per tipand pelleted by centrifuging at 1000 rpm for 5 minutes at 4° C.,followed by washing again with ½ volume of PBS recentrifugation. Thepellets were washed a third time, the suspended cells collected andwashed 2× with PBS. The cells were then suspended into 10 ml PBS. BufferC1 was equilibrated at 4° C. Qiagen protease #19155 was diluted into6.25 ml cold ddH₂O to a final concentration of 20 mg/ml and equilibratedat 4° C. 10 ml of G2 Buffer was prepared by diluting Qiagen RNAse Astock (100 mg/ml) to a final concentration of 200 μg/ml.

Buffer C1 (10 ml, 4° C.) and ddH2O (40 ml, 4° C.) were then added to the10 ml of cell suspension, mixed by inverting and incubated on ice for 10minutes. The cell nuclei were pelleted by centrifuging in a Beckmanswinging bucket rotor at 2500 rpm at 4° C. for 15 minutes. Thesupernatant was discarded and the nuclei were suspended with a vortexinto 2 ml Buffer C1 (at 4° C.) and 6 ml ddH₂O, followed by a second 4°C. centrifugation at 2500 rpm for 15 minutes. The nuclei were thenresuspended into the residual buffer using 200 μl per tip. G2 buffer (10ml) was added to the suspended nuclei while gentle vortexing wasapplied. Upon completion of buffer addition, vigorous vortexing wasapplied for 30 seconds. Quiagen protease (200 μl, prepared as indicatedabove) was added and incubated at 50° C. for 60 minutes. The incubationand centrifugation was repeated until the lysates were clear (e.g.,incubating additional 30-60 minutes, pelleting at 3000× g for 10 min.,4° C.).

Solid Human Tumor Sample Preparation and Lysis

Tumor samples were weighed and placed into 50 ml conical tubes and heldon ice. Processing was limited to no more than 250 mg tissue perpreparation (1 tip/preparation). The protease solution was freshlyprepared by diluting into 6.25 ml cold ddH₂O to a final concentration of20 mg/ml and stored at 4° C. G2 buffer (20 ml) was prepared by dilutingDNAse A to a final concentration of 200 mg/ml (from 100 mg/ml stock).The tumor tissue was homogenated in 19 ml G2 buffer for 60 seconds usingthe large tip of the polytron in a laminar-flow TC hood in order toavoid inhalation of aerosols, and held at room temperature. Betweensamples, the polytron was cleaned by spinning at 2×30 seconds each in 2LddH₂O, followed by G2 buffer (50 ml). If tissue was still present on thegenerator tip, the apparatus was disassembled and cleaned.

Quiagen protease (prepared as indicated above, 1.0 ml) was added,followed by vortexing and incubation at 50° C. for 3 hours. Theincubation and centrifugation was repeated until the lysates were clear(e.g., incubating additional 30-60 minutes, pelleting at 3000× g for 10min., 4° C.).

Human Blood Preparation and Lysis

Blood was drawn from healthy volunteers using standard infectious agentprotocols and citrated into 10 ml samples per tip. Quiagen protease wasfreshly prepared by dilution into 6.25 ml cold ddH₂O to a finalconcentration of 20 mg/ml and stored at 4° C. G2 buffer was prepared bydiluting RNAse A to a final concentration of 200 μg/ml from 100 mg/mlstock. The blood (10 ml) was placed into a 50 ml conical tube and 10 mlC1 buffer and 30 ml ddH₂O (both previously equilibrated to 4° C.) wereadded, and the components mixed by inverting and held on ice for 10minutes. The nuclei were pelleted with a Beckman swinging bucket rotorat 2500 rpm, 4° C. for 15 minutes and the supernatant discarded. With avortex, the nuclei were suspended into 2 ml C1 buffer (4° C.) and 6 mlddH₂O (4° C.). Vortexing was repeated until the pellet was white. Thenuclei were then suspended into the residual buffer using a 200 μl tip.G2 buffer (10 ml) were added to the suspended nuclei while gentlyvortexing, followed by vigorous vortexing for 30 seconds. Quiagenprotease was added (200 μl) and incubated at 50° C. for 60 minutes. Theincubation and centrifugation was repeated until the lysates were clear(e.g., incubating additional 30-60 minutes, pelleting at 3000× g for 10min., 4° C.).

Purification of Cleared Lysates

(1) Isolation of Genomic DNA

Genomic DNA was equilibrated (I sample per maxi tip preparation) with 10ml QBT buffer. QF elution buffer was equilibrated at 50° C. The sampleswere vortexed for 30 seconds, then loaded onto equilibrated tips anddrained by gravity. The tips were washed with 2×15 ml QC buffer. The DNAwas eluted into 30 ml silanized, autoclaved 30 ml Corex tubes with 15 mlQF buffer (50° C.). Isopropanol (10.5 ml) was added to each sample, thetubes covered with parafin and mixed by repeated inversion until the DNAprecipitated. Samples were pelleted by centrifugation in the SS-34 rotorat 15,000 rpm for 10 minutes at 4° C. The pellet location was marked,the supernatant discarded, and 10 ml 70% ethanol (4° C.) was added.Samples were pelleted again by centrifugation on the SS-34 rotor at10,000 rpm for 10 minutes at 4° C. The pellet location was marked andthe supernatant discarded. The tubes were then placed on their side in adrying rack and dried 10 minutes at 37° C., taking care not to overdrythe samples.

After drying, the pellets were dissolved into 1.0 ml TE (pH 8.5) andplaced at 50° C. for 1-2 hours. Samples were held overnight at 4° C. asdissolution continued. The DNA solution was then transferred to 1.5 mltubes with a 26 gauge needle on a tuberculin syringe. The transfer wasrepeated 5× in order to shear the DNA. Samples were then placed at 50°C. for 1-2 hours.

(2) Quantitation of Genomic DNA and Preparation for Gene AmplificationAssay

The DNA levels in each tube were quantified by standard A₂₆₀, A₂₈₀spectrophotometry on a 1:20 dilution (5 μl DNA+95 μl ddH₂O) using the0.1 ml quartz cuvetts in the Beckman DU640 spectrophotometer. A₂₆₀/A₂₈₀ratios were in the range of 1.8-1.9. Each DNA samples was then dilutedfurther to approximately 200 ng/ml in TE (pH 8.5). If the originalmaterial was highly concentrated (about 700 ng/μl), the material wasplaced at 50° C. for several hours until resuspended.

Fluorometric DNA quantitation was then performed on the diluted material(20-600 ng/ml) using the manufacturer's guidelines as modified below.This was accomplished by allowing a Hoeffer DyNA Quant 200 fluorometerto warm-up for about 15 minutes. The Hoechst dye working solution(#H33258, 10 μl, prepared within 12 hours of use) was diluted into 100ml 1× TNE buffer. A 2 ml cuvette was filled with the fluorometersolution, placed into the machine, and the machine was zeroed. pGEM3Zf(+) (2 μl, lot #360851026) was added to 2 ml of fluorometer solutionand calibrated at 200 units. An additional 2 μl of pGEM 3Zf(+) DNA wasthen tested and the reading confirmed at 400+/−10 units. Each sample wasthen read at least in triplicate. When 3 samples were found to be within10% of each other, their average was taken and this value was used asthe quantification value.

The fluorometricly determined concentration was then used to dilute eachsample to 10 ng/μl in ddH₂O. This was done simultaneously on alltemplate samples for a single TaqMan plate assay, and with enoughmaterial to run 500-1000 assays. The samples were tested in triplicatewith Taqman™ primers and probe both B-actin and GAPDH on a single platewith normal human DNA and no-template controls. The diluted samples wereused provided that the CT value of normal human DNA subtracted from testDNA was +/−1 Ct. The diluted, lot-qualified genomic DNA was stored in1.0 ml aliquots at −80° C. Aliquots which were subsequently to be usedin the gene amplification assay were stored at 4° C. Each 1 ml aliquotis enough for 8-9 plates or 64 tests.

Gene Amplification Assay

The PRO polypeptide compounds of the invention were screened in thefollowing primary tumors and the resulting ΔCt values greater than orequal to 1.0 are reported in Table 9 below.

TABLE 9 ΔCt values in lung and colon primary tumors and cell line modelsPrimary Tumors or Cell PRO lines 187 PRO533 PRO214 PRO343 PRO211 PRO230PRO246 PRO317 PRO232 PRO269 PRO304 PRO339 LT7 1.52 1.04 1.08 LT13 2.741.85 2.71 1.88 3.42 1.63 1.90 1.27 1.29 1.04 2.98 1.83 2.23 2.26 3.221.68 2.24 2.44 2.84 2.93 2.15 2.75 2.53 1.82 LT3 1.57 1.97 1.06 1.861.17 LT4 1.17 1.18 LT9 1.42 1.04 1.08 1.03 LT12 2.70 1.38 2.23 1.51 2.861.54 2.54 2.40 1.14 1.15 1.26 2.90 1.49 1.50 1.27 2.96 2.47 1.74 2.272.92 1.25 2.68 2.28 1.34 LT30 1.67 2.13 1.36 LT21 1.26 1.09 1.50 LT1-a1.02 1.18 1.29 LT6 1.93 LT10 1.96 1.07 2.57 LT11 1.09 1.67 1.00 2.051.32 3.43 2.20 1.14 1.51 1.39 1.80 1.89 1.14 1.41 2.33 1.54 1.02 LT153.75 1.77 3.62 2.44 4.32 2.11 2.06 1.86 1.36 1.34 3.92 1.58 1.30 2.164.47 1.56 2.76 3.49 3.64 1.63 2.94 3.56 3.32 2.68 LT16 2.10 1.66 1.701.25 1.15 1.55 1.00 2.04 1.08 1.83 1.33 LT17 1.32 1.93 1.15 1.85 1.262.68 2.29 1.35 1.42 1.68 1.63 1.87 2.30 1.39 1.69 2.03 1.30 1.10 1.331.30 LT18 1.17 1.04 LT19 4.05 1.67 2.09 3.82 2.42 4.05 1.91 2.51 1.211.60 1.15 3.99 1.98 2.55 4.92 1.68 2.03 4.93 1.16 3.78 4.76 HF- 1.58000840 Calu-1 1.08 SW900 1.86 CT2 3.56 2.49 1.95 1.42 2.75 3.49 2.36 CT32.06 1.15 1.34 CT8 1.01 1.48 1.29 1.58 CT10 1.81 1.84 1.88 1.00 1.881.49 1.55 CT12 1.81 1.74 1.13 CT14 1.82 2.48 2.33 1.36 1.72 1.24 CT151.63 2.06 1.33 1.41 1.04 CT16 1.95 1.78 1.40 CT17 2.04 2.40 1.74 CT11.24 1.22 1.27 1.25 2.41 1.34 1.46 1.14 CT4 1.36 1.77 1.33 1.32 1.101.17 2.05 1.42 1.02 CT5 2.96 1.56 2.68 1.76 2.27 1.33 1.59 2.99 2.761.64 2.39 CT6 1.10 1.33 1.01 1.14 CT7 1.40 1.66 1.39 1.00 CT9 1.39 1.161.09 1.24 1.13 CT11 2.22 2.05 1.55 2.01 1.75 1.48 1.92 2.26 1.85 1.831.12 HF000539 1.57 SW620 1.14 HF000611 4.64 HF000733 1.93 2.33 HF0007161.68 2.82 CT18 1.29Summary

Because amplification of the various DNA's as described above occurs invarious tumors, it is likely associated with tumor formation and/orgrowth. As a result, antagonists (e.g., antibodies) directed againstthese polypeptides would be expected to be useful in cancer therapy.

Example 94 Detection of PRO Polypeptides that Affect Glucose or FFAUptake by Primary Rat Adipocytes (Assay 94)

This assay is designed to determine whether PRO polypeptides show theability to affect glucose or FFA uptake by adipocyte cells. PROpolypeptides testing positive in this assay would be expected to beuseful for the therapeutic treatment of disorders where either thestimulation or inhibition of glucose uptake by adipocytes would bebeneficial including, for example, obesity, diabetes or hyper- orhypo-insulinemia.

In a 96 well format, PRO polypeptides to be assayed are added to primaryrat adipocytes, and allowed to incubate overnight. Samples are taken at4 and 16 hours and assayed for glycerol, glucose and FFA uptake. Afterthe 16 hour incubation, insulin is added to the media and allowed toincubate for 4 hours. At this time, a sample is taken and glycerol,glucose and FFA uptake is measured. Media containing insulin without thePRO polypeptide is used as a positive reference control. As the PROpolypeptide being tested may either stimulate or inhibit glucose and FFAuptake, results are scored as positive in the assay if greater than 1.5times or less than 0.5 times the insulin control.

The following PRO polypeptides tested positive as stimulators of glucoseand/or FFA uptake in this assay: PRO221, PRO235, PRO245, PRO295, PRO301and PRO332.

The following PRO polypeptides tested positive as inhibitors of glucoseand/or FFA uptake in this assay: PRO214, PRO219, PRO228, PRO222, PRO231and PRO265.

Example 95 Chondrocyte Re-differentiation Assay (Assay 110)

This assay shows that certain polypeptides of the invention act toinduce redifferentiation of chondrocytes, therefore, are expected to beuseful for the treatment of various bone and/or cartilage disorders suchas, for example, sports injuries and arthritis. The assay is performedas follows. Porcine chondrocytes are isolated by overnight collagenasedigestion of articulary cartilage of metacarpophalangeal joints of 4-6month old female pigs. The isolated cells are then seeded at 25,000cells/cm² in Ham F-12 containing 10% FBS and 4 μg/ml gentamycin. Theculture media is changed every third day and the cells are then seededin 96 well plates at 5,000 cells/well in 100 μl of the same mediawithout serum and 100 μl of the test PRO polypeptide, 5 nM staurosporin(positive control) or medium alone (negative control) is added to give afinal volume of 200 L/well. After 5 days of incubation at 37° C., apicture of each well is taken and the differentiation state of thechondrocytes is determined. A positive result in the assay occurs whenthe redifferentiation of the chondrocytes is determined to be moresimilar to the positive control than the negative control.

The following polypeptide tested positive in this assay: PRO214, PRO219,PRO229, PRO222, PRO224, PRO230, PRO257, PRO272 and PRO301.

Example 96 Fetal Hemoglobin Induction in an Erythroblastic Cell Line(Assay 107)

This assay is useful for screening PRO polypeptides for the ability toinduce the switch from adult hemoglobin to fetal hemoglobin in anerythroblastic cell line. Molecules testing positive in this assay areexpected to be useful for therapeutically treating various mammalianhemoglobin-associated disorders such as the various thalassemias. Theassay is performed as follows. Erythroblastic cells are plated instandard growth medium at 1000 cells/well in a 96 well format. PROpolypeptides are added to the growth medium at a concentration of 0.2%or 2% and the cells are incubated for 5 days at 37° C. As a positivecontrol, cells are treated with 100 μM hemin and as a negative control,the cells are untreated. After 5 days, cell lysates are prepared andanalyzed for the expression of gamma globin (a fetal marker). A positivein the assay is a gamma globin level at least 2-fold above the negativecontrol.

The following polypeptides tested positive in this assay: PRO221 andPRO245.

Example 97 Mouse Kidney Mesangial Cell Proliferation Assay (Assay 92)

This assay shows that certain polypeptides of the invention act toinduce proliferation of mammalian kidney mesangial cells and, therefore,are useful for treating kidney disorders associated with decreasedmesangial cell function such as Berger disease or other nephropathiesassociated with Schönlein-Henoch purpura, celiac disease, dermatitisherpetiformis or Crohn disease. The assay is performed as follows. Onday one, mouse kidney mesangial cells are plated on a 96 well plate ingrowth media (3:1 mixture of Dulbecco's modified Eagle's medium andHam's F12 medium, 95% fetal bovine serum, 5% supplemented with 14 mMHEPES) and grown overnight. On day 2, PRO polypeptides are diluted at 2concentrations(1% and 0.1%) in serum-free medium and added to the cells.Control samples are serum-free medium alone. On day 4, 20 μl of the CellTiter 96 Aqueous one solution reagent (Progema) was added to each welland the colormetric reaction was allowed to proceed for 2 hours. Theabsorbance (OD) is then measured at 490 nm. A positive in the assay isanything that gives an absorbance reading which is at least 15% abovethe control reading.

The following polypeptide tested positive in this assay: PRO227.

Example 98 Proliferation of Rat Utricular Supporting Cells (Assay 54)

This assay shows that certain polypeptides of the invention act aspotent mitogens for inner ear supporting cells which are auditory haircell progenitors and, therefore, are useful for inducing theregeneration of auditory hair cells and treating hearing loss inmammals. The assay is performed as follows. Rat UEC-4 utricularepithelial cells are aliquoted into 96 well plates with a density of3000 cells/well in 200 μl of serum-containing medium at 33° C. The cellsare cultured overnight and are then switched to serum-free medium at 37°C. Various dilutions of PRO polypeptides (or nothing for a control) arethen added to the cultures and the cells are incubated for 24 hours.After the 24 hour incubation, ³H-thymidine (1 μCi/well) is added and thecells are then cultured for an additional 24 hours. The cultures arethen washed to remove unincorporated radiolabel, the cells harvested andCpm per well determined. Cpm of at least 30% or greater in the PROpolypeptide treated cultures as compared to the control cultures isconsidered a positive in the assay.

The following polypeptides tested positive in this assay: PRO310 andPRO346.

Example 99 Chondrocyte Proliferation Assay (Assay 111)

This assay is designed to determine whether PRO polypeptides of thepresent invention show the ability to induce the proliferation and/orredifferentiation of chondrocytes in culture. PRO polypeptides testingpositive in this assay would be expected to be useful for thetherapeutic treatment of various bone and/or cartilage disorders suchas, for example, sports injuries and arthritis.

Porcine chondrocytes are isolated by overnight collagenase digestion ofarticular cartilage of the metacarpophalangeal joint of 4-6 month oldfemale pigs. The isolated cells are then seeded at 25,000 cells/cm² inHam F-12 containing 10% FBS and 4 μg/ml gentamycin. The culture media ischanged every third day and the cells are reseeded to 25,000 cells/cm²every five days. On day 12, the cells are seeded in 96 well plates at5,000 cells/well in 100 μl of the same media without serum and 100 μl ofeither serum-free medium (negative control), staurosporin (finalconcentration of 5 nM; positive control) or the test PRO polypeptide areadded to give a final volume of 200 μl/well. After 5 days at 37° C., 20μl of Alamar blue is added to each well and the plates are incubated foran additional 3 hours at 37° C. The fluorescence is then measured ineach well (Ex:530 nm; Em: 590 nm). The fluorescence of a platecontaining 200 μl of the serum-free medium is measured to obtain thebackground. A positive result in the assay is obtained when thefluorescence of the PRO polypeptide treated sample is more like that ofthe positive control than the negative control.

The following PRO polypeptides tested positive in this assay: PRO219,PRO222, PRO317, PRO257, PRO265, PRO287, PRO272 and PRO533.

Example 100 Inhibition of Heart Neonatal Hypertrophy Induced by LIF+ET-1(Assay 74)

This assay is designed to determine whether PRO polypeptides of thepresent invention show the ability to inhibit neonatal heart hypertrophyinduced by LIF and endothelin-1 (ET-1). A test compound that provides apositive response in the present assay would be useful for thetherapeutic treatment of cardiac insufficiency diseases or disorderscharacterized or associated with an undesired hypertrophy of the cardiacmuscle.

Cardiac myocytes from 1-day old Harlan Sprague Dawley rats (180 μl at7.5×10⁴/ml, serum<0.1, freshly isolated) are introduced on day 1 to96-well plates previously coated with DMEM/F12+4%FCS. Test PROpolypeptide samples or growth medium alone (negative control) are thenadded directly to the wells on day 2 in 20 μl volume. LIF+ET-1 are thenadded to the wells on day 3. The cells are stained after an additional 2days in culture and are then scored visually the next day. A positive inthe assay occurs when the PRO polypeptide treated myocytes are visuallysmaller on the average or less numerous than the untreated myocytes.

The following PRO polypeptides tested positive in this assay: PRO238.

Example 101 Tissue Expression Distribution

Oligonucleotide probes were constructed from some of the PROpolypeptide-encoding nucleotide sequences shown in the accompanyingfigures for use in quantitative PCR amplification reactions. Theoligonucleotide probes were chosen so as to give an approximately200-600 base pair amplified fragment from the 3′ end of its associatedtemplate in a standard PCR reaction. The oligonucleotide probes wereemployed in standard quantitative PCR amplification reactions with cDNAlibraries isolated from different human adult and/or fetal tissuesources and analyzed by agarose gel electrophoresis so as to obtain aquantitative determination of the level of expression of the PROpolypeptide-encoding nucleic acid in the various tissues tested.Knowledge of the expression pattern or the differential expression ofthe PRO polypeptide-encoding nucleic acid in various different humantissue types provides a diagnostic marker useful for tissue typing, withor without other tissue-specific markers, for determining the primarytissue source of a metastatic tumor, and the like. These assays providedthe following results.

DNA Molecule Tissues With Significant Expression Tissues LackingSignificant Expression DNA34436-1238 lung, placenta, brain testisDNA35557-1137 lung, kidney, brain placenta DNA35599-1168 kidney, brainliver, placenta DNA35668-1171 liver, lung, kidney placenta, brainDNA36992-1168 liver, lung, kidney, brain placenta DNA39423-1182 kidney,brain liver DNA40603-1232 liver brain, kidney, lung DNA40604-1187 liverbrain, kidney, lung DNA41379-1236 lung, brain liver DNA33206-1165 heart,spleen, dendrocytes substantia nigra, hippocampus, cartilage, prostate,HUVEC DNA34431-1177 spleen, HUVEC, cartilage, heart, uterus brain, colontumor, prostate, THP-1 macrophages DNA41225-1217 HUVEC, uterus, colontumor, cartilage, spleen, brain, heart, IM-9 lymphoblasts prostate

Example 102 In situ Hybridization

In situ hybridization is a powerful and versatile technique for thedetection and localization of nucleic acid sequences within cell ortissue preparations. It may be useful, for example, to identify sites ofgene expression, analyze the tissue distribution of transcription,identify and localize viral infection, follow changes in specific mRNAsynthesis and aid in chromosome mapping.

In situ hybridization was performed following an optimized version ofthe protocol by Lu and Gillett, Cell Vision 1:169-176 (1994), usingPCR-generated ³³P-labeled riboprobes. Briefly, formalin-fixed,paraffin-embedded human tissues were sectioned, deparaffinized,deproteinated in proteinase K (20 g/ml) for 15 minutes at 37° C., andfurther processed for in situ hybridization as described by Lu andGillett, supra. A [33-P] UTP-labeled antisense riboprobe was generatedfrom a PCR product and hybridized at 55° C. overnight. The slides weredipped in Kodak NTB2 nuclear track emulsion and exposed for 4 weeks.

³³P-Riboprobe Synthesis

6.0 μl (125 mCi) of ³³P-UTP (Amersham BF 1002, SA<2000 Ci/mmol) werespeed vac dried. To each tube containing dried ³³P-UTP, the followingingredients were added:

-   -   2.0 μl 5× transcription buffer    -   1.0 μl DTT (100 mM)    -   2.0 μl NTP mix (2.5 mM: 10 μ; each of 10 mM GTP, CTP & ATP+10 μl        H₂O)    -   1.0 UTP (50 μM)    -   1.0 μl Rnasin    -   1.0 μl DNA template (1 μg)    -   1.0 μl H₂O    -   1.0 μl RNA polymerase (for PCR products T3 AS, T7=S, usually)

The tubes were incubated at 37° C. for one hour. 1.0 μl RQ1 DNase wereadded, followed by incubation at 37° C. for 15 minutes. 90 μl TE (10 mMTris pH 7.6/1 mM EDTA pH 8.0) were added, and the mixture was pipettedonto DE81 paper. The remaining solution was loaded in a Microcon-50ultrafiltration unit, and spun using program 10 (6 minutes). Thefiltration unit was inverted over a second tube and spun using program 2(3 minutes). After the final recovery spin, 100 μl TE were added. 1 μlof the final product was pipetted on DE81 paper and counted in 6 ml ofBiofluor II.

The probe was run on a TBE/urea gel. 1-3 μl of the probe or 5 al of RNAMrk III were added to 3 μl of loading buffer. After heating on a 95° C.heat block for three minutes, the gel was immediately placed on ice. Thewells of gel were flushed, the sample loaded, and run at 180-250 voltsfor 45 minutes. The gel was wrapped in saran wrap and exposed to XARfilm with an intensifying screen in −70° C. freezer one hour toovernight.

³³P-Hybridization

A. Pretreatment of Frozen Sections

The slides were removed from the freezer, placed on aluminium trays andthawed at room temperature for 5 minutes. The trays were placed in 55°C. incubator for five minutes to reduce condensation. The slides werefixed for 10 minutes in 4% paraformaldehyde on ice in the fume hood, andwashed in 0.5×SSC for 5 minutes, at room temperature (25 ml 20×SSC+975ml SQ H₂O). After deproteination in 0.5 μg/ml proteinase K for 10minutes at 37° C. (12.5 μl of 10 mg/ml stock in 250 ml prewarmedRNase-free RNAse buffer), the sections were washed in 0.5×SSC for 10minutes at room temperature. The sections were dehydrated in 70%, 95%,100% ethanol, 2 minutes each.

B. Pretreatment of Paraffin-embedded Sections

The slides were deparaffinized, placed in SQ H₂O, and rinsed twice in2×SSC at room temperature, for 5 minutes each time. The sections weredeproteinated in 20 μg/ml proteinase K (500 μl of 10 mg/ml in 250 mlRNase-free RNase buffer; 37° C., 15 minutes)—human embryo, or 8×proteinase K (100 μl in 250 ml Rnase buffer, 37° C., 30minutes)—formalin tissues. Subsequent rinsing in 0.5×SSC and dehydrationwere performed as described above.

C. Prehybridization

The slides were laid out in a plastic box lined with Box buffer (4×SSC,50% formamide)—saturated filter paper. The tissue was covered with 50 μlof hybridization buffer (3.75 g Dextran Sulfate+6 ml SQ H₂O), vortexedand heated in the microwave for 2 minutes with the cap loosened. Aftercooling on ice, 18.75 ml formamide, 3.75 ml 20×SSC and 9 ml SQ H₂O wereadded, the tissue was vortexed well, and incubated at 42° C. for 1-4hours.

D. Hybridization

1.0×10⁶ cpm probe and 1.0 μl tRNA (50 mg/ml stock) per slide were heatedat 95° C. for 3 minutes. The slides were cooled on ice, and 48 μlhybridization buffer were added per slide. After vortexing, 50 μl ³³Pmix were added to 50 μl prehybridization on slide. The slides wereincubated overnight at 55° C.

E. Washes

Washing was done 2×10 minutes with 2×SSC, EDTA at room temperature (400ml 20×SSC+16 ml 0.25M EDTA, V_(f)=4L), followed by RNaseA treatment at37° C. for 30 minutes (500 μl of 10 mg/ml in 250 ml Rnase buffer=20μg/ml), The slides were washed 2×10 minutes with 2×SSC, EDTA at roomtemperature. The stringency wash conditions were as follows: 2 hours at55° C., 0.1×SSC, EDTA (20 ml 20×SSC+16 ml EDTA, V_(f)=4L).

F. Oligonucleotides

In situ analysis was performed on a variety of DNA sequences disclosedherein. The oligonucleotides employed for these analyses are as follows.

(1) DNA33094-1131 (PRO217) p15′-GGATTCTAATACGACTCACTATAGGGCTCAGAAAAGCGCAACAGAGAA-3′ (SEQ ID NO:348)p2 5′-CTATGAAATTAACCCTCACTAAAGGGATGTCTTCCATGCCAACCTTC-3′ (SEQ ID NO:349)(2) DNA33223-1136 (PRO230) p15′-GGATTCTAATACGACTCACTATAGGGCGGCGATGTCCACTGGGGCTAC-3′ (SEQ ID NO:350)p2 5′-CTATGAAATTAACCCTCACTAAAGGGACGAGGAAGATGGGCGGATGGT-3′ (SEQ IDNO:351) (3) DNA34435-1140 (PRO232) p15′-GGATTCTAATACGACTCACTATAGGGCACCCACGCGTCCGGCTGCTT-3′ (SEQ ID NO:352) p25′-CTATGAAATTAACCCTCACTAAAGGGACGGGGGACACCACGGACCAGA-3′ (SEQ ID NO:353)(4) DNA35639-1172 (PRO246) p15′-GGATTCTAATACGACTCACTATAGGGCTTGCTGCGGTTTTTGTTCCTG-3′ (SEQ ID NO:354)p2 5′-CTATGAAATTAACCCTCACTAAAGGGAGCTGCCGATCCCACTGGTATT-3′ (SEQ IDNO:355) (5) DNA49435-1219 (PRO533) p15′-GGATTCTAATACGACTCACTATAGGGCGGATCCTGGCCGGCCTCTG-3′ (SEQ ID NO:356) p25′-CTATGAAATTAACCCTCACTAAAGGGAGCCCGGGCATGGTCTCAGTTA-3′ (SEQ ID NO:357)(6) DNA35638-1141 (PRO245) p15′-GGATTCTAATACGACTCACTATAGGGCGGGAAGATGGCGAGGAGGAG-3′ (SEQ ID NO:358) p25′-CTATGAAATTAACCCTCACTAAAGGGACCAAGGCCACAAACGGAAATC-3′ (SEQ ID NO:359)(7) DNA33089-1132 (PRO221) p15′-GGATTCTAATACGACTCACTATAGGGCTGTGCTTTCATTCTGCCAGTA-3′ (SEQ ID NO:360)p2 5′-CTATGAAATTAACCCTCACTAAAGGGAGGGTACAATTAAGGGGTGGAT-3′ (SEQ IDNO:361) (8) DNA35918-1174 (PRO258) p15′-GGATTCTAATACGACTCACTATAGGGCCCGCCTCGCTCCTGCTCCTG-3′ (SEQ ID NO:362) p25′-CTATGAAATTAACCCTCACTAAAGGGAGGATTGCCGCGACCCTCACAG-3′ (SEQ ID NO:363)(9) DNA32286-1191 (PRO214) p15′-GGATTCTAATACGACTCACTATAGGGCCCCTCCTGCCTTCCCTGTCC-3′ (SEQ ID NO:364) p25′-CTATGAAATTAACCCTCACTAAAGGGAGTGGTGGCCGCGATTATCTGC-3′ (SEQ ID NO:365)(10) DNA33221-1133 (PRO224) p15′-GGATTCTAATACGACTCACTATAGGGCGCAGCGATGGCAGCGATGAGG-3′ (SEQ ID NO:366)p2 5′-CTATGAAATTAACCCTCACTAAAGGGACAGACGGGGCAGAGGGAGTG-3′ (SEQ ID NO:367)(11) DNA35557-1137 (PRO234) p15′-GGATTCTAATACGACTCACTATAGGGCCAGGAGGCGTGAGGAGAAAC-3′ (SEQ ID NO:368) p25′-CTATGAAATTAACCCTCACTAAAGGGAAAGACATGTCATCGGGAGTGG-3′ (SEQ ID NO:369)(12) DNA33100-1159 (PRO229) p15′-GGATTCTAATACGACTCACTATAGGGCCGGGTGGAGGTGGAACAGAAA-3′ (SEQ ID NO:370)p2 5′-CTATGAAATTAACCCTCACTAAAGGGACACAGACAGAGCCCCATACGC-3′ (SEQ IDNO:371) (13) DNA34431-1177 (PRO263) p15′-GGATTCTAATACGACTCACTATAGGGCCAGGGAAATCCGGATGTCTC-3′ (SEQ ID NO:372) p25′-CTATGAAATTAACCCTCACTAAAGGGAGTAAGGGGATGCCACCGAGTA-3′ (SEQ ID NO:373)(14) DNA38268-1188 (PRO295) p15′-GGATTCTAATACGACTCACTATAGGGCCAGCTACCCGCAGGAGGAGG-3′ (SEQ ID NO:374) p25′-CTATGAAATTAACCCTCACTAAAGGGATCCCAGGTGATGAGGTCCAGA-3′ (SEQ ID NO:375)

G. Results

In situ analysis was performed on a variety of DNA sequences disclosedherein. The results from these analyses are as follows.

(1) DNA33094-1131 (PRO217)

Highly distinctive expression pattern, that does not indicate an obviousbiological function. In the human embryo it was expressed in outersmooth muscle layer of the GI tract, respiratiry cartilage, branchingrespiratory epithelium, osteoblasts, tendons, gonad, in the optic nervehead and developing dermis. In the adult expression was observed in theepidermal pegs of the chimp tongue, the basal epithelial/myoepithelialcells of the prostate and urinary bladder. Also expressed in thealveolar lining cells of the adult lung, mesenchymal cells juxtaposed toerectile tissue in the penis and the cerebral cortex (probably glialcells). In the kidney, expression was only seen in disease, in cellssurrounding thyroidized renal tubules.

-   Human fetal tissues examined (E12-E16 weeks) include: Placenta,    umbilical cord, liver, kidney, adrenals, thyroid, lungs, heart,    great vessels, oesophagus, stomach, small intestine, spleen, thymus,    pancreas, brain, eye, spinal cord, body wall, pelvis and lower limb.-   Adult human tissues examined: Kidney (normal and end-stage),    adrenal, myocardium, aorta, spleen, lymph node, gall bladder,    pancreas, lung, skin, eye (inc. retina), prostate, bladder, liver    (normal, cirrhotic, acute failure).-   Non-human primate tissues examined:    -   (a) Chimp Tissues: Salivary gland, stomach, thyroid,        parathyroid, skin, thymus, ovary, lymph node.    -   (b) Rhesus Monkey Tissues: Cerebral cortex, hippocampus,        cerebellum, penis.        (2) DNA33223-1136 (PRO230)

Sections show an intense signal associated with arterial and venousvessels in the fetus. In arteries the signal appeared to be confined tosmooth-muscle/pericytic cells. The signal is also seen in capillaryvessels and in glomeruli. It is not clear whether or not endothelialcells are expressing this mRNA. Expression is also observed inepithelial cells in the fetal lens. Strong expression was also seen incells within placental trophoblastic villi, these cells lie between thetrophoblast and the fibroblast-like cells that express HGF—uncertainhistogenesis. In the adult, there was no evidence of expression and thewall of the aorta and most vessels appear to be negative. However,expression was seen over vascular channels in the normal prostate and inthe epithelium lining the gallbladder. Insurers expression was seen inthe vessels of the soft-tissue sarcoma and a renal cell carcinoma. Insummary, this is a molecule that shows relatively specific vascularexpression in the fetus as well as in some adult organs. Expression wasalso observed in the fetal lens and the adult gallbladder.

In a secondary screen, vascular expression was observed, similar to thatobserved above, seen in fetal blocks. Expression is on vascular smoothmuscle, rather than endothelium. Expression also seen in smooth muscleof the developing oesophagus, so as reported previously, this moleculeis not vascular specific. Expression was examined in 4 lung and 4 breastcarcinomas. Substantial expression was seen in vascular smooth muscle ofat least 3/4 lung cancers and 2/4 breast cancers. In addition, in onebreast carcinoma, expression was observed in peritumoral stromal cellsof uncertain histogenesis (possibly myofibroblasts). No endothelial cellexpression was observed in this study.

(3) DNA34435-1140 (PRO232)

Strong expression in prostatic epithelium and bladder epithelium, lowerlevel of expression in bronchial epithelium. High background/low levelexpression seen in a number of sites, including among others, bone,blood, chondrosarcoma, adult heart and fetal liver. It is felt that thislevel of signal represents background, partly because signal at thislevel was seen over the blood. All other tissues negative.

-   Human fetal tissues examined (E12-E16 weeks) include: Placenta,    umbilical cord, liver, kidney, adrenals, thyroid, lungs, heart,    great vessels, oesophagus, stomach, small intestine, spleen, thymus,    pancreas, brain, eye, spinal cord, body wall, pelvis, testis and    lower limb.-   Adult human tissues examined: Kidney (normal and end-stage),    adrenal, spleen, lymph node, pancreas, lung, eye (inc. retina),    bladder, liver (normal, cirrhotic, acute failure).-   Non-human primate tissues examined:    -   Chimp Tissues: adrenal    -   Rhesus Monkey Tissues: Cerebral cortex, hippocampus

In a secondary screen, expression was observed in the epithelium of theprostate, the superficial layers of the urethelium of the urinarybladder, the urethelium lining the renal pelvis and the urethelium ofthe ureter (1 out of 2 experiments). The urethra of a rhesus monkey wasnegative; it is unclear whether this represents a true lack ofexpression by the urethra, or if it is the result of a failure of theprobe to cross react with rhesus tissue. The findings in the prostateand bladder are similar to those previously described using an isotopicdetection technique. Expression of the mRNA for this antigen is NOTprostate epithelial specific. The antigen may serve as a useful markerfor urethelial derived tissues. Expression in the superficial,post-mitotic cells, of the urinary tract epithelium also suggest that itis unlikely to represent a specific stem cell marker, as this would beexpected to be expressed specifically in basal epithelium.

(4) DNA35639-1172 (PRO246)

Strongly expressed in fetal vascular endothelium, including tissues ofthe CNS. Lower level of expression in adult vasculature, including theCNS. Not obviously expressed at higher levels in tumor vascularendothelium. Signal also seen over bone matrix and adult spleen, notobviously cell associated, probably related to non-specific backgroundat these sites.

-   Human fetal tissues examined (E12-E16 weeks) include: Placenta,    umbilical cord, liver, kidney, adrenals, thyroid, lungs, heart,    great vessels, oesophagus, stomach, small intestine, spleen, thymus,    pancreas, brain, eye, spinal cord, body wall, pelvis, testis and    lower limb.-   Adult human tissues examined: Kidney (normal and end-stage),    adrenal, spleen, lymph node, pancreas, lung, eye (inc. retina),    bladder, liver (normal, cirrhotic, acute failure).-   Non-human primate tissues examined:    -   Chimp Tissues: adrenal    -   Rhesus Monkey Tissues: Cerebral cortex, hippocampus        (5) DNA49435-1219 (PRO533)

Moderate expression over cortical neurones in the fetal brain.Expression over the inner aspect of the fetal retina, possibleexpression in the developing lens. Expression over fetal skin,cartilage, small intestine, placental villi and umbilical cord. In adulttissues there is an extremely high level of expression over thegallbladder epithelium. Moderate expression over the adult kidney,gastric and colonic epithelia. Low-level expression was observed overmany cell types in many tissues, this may be related to stickiness ofthe probe, these data should therefore be interpreted with a degree ofcaution.

-   Human fetal tissues examined (E12-E16 weeks) include: Placenta,    umbilical cord, liver, kidney, adrenals, thyroid, lungs, heart,    great vessels, oesophagus, stomach, small intestine, spleen, thymus,    pancreas, brain, eye, spinal cord, body wall, pelvis, testis and    lower limb.-   Adult human tissues examined: Kidney (normal and end-stage),    adrenal, spleen, lymph node, pancreas, lung, eye (inc. retina),    bladder, liver (normal, cirrhotic, acute failure).-   Non-human primate tissues examined:    -   Chimp Tissues: adrenal    -   Rhesus Monkey Tissues: Cerebral cortex, hippocampus, cerebellum.        (6) DNA35638-1141 (PRO245)

Expression observed in the endothelium lining a subset of fetal andplacental vessels. Endothelial expression was confined to these tissueblocks. Expression also observed over intermediate trophoblast cells ofplacenta. Expression also observed tumor vasculature but not in thevasculature of normal tissues of the same type. All other tissuesnegative.

-   Fetal tissues examined (E12-E16 weeks) include: Placenta, umbilical    cord, liver, kidney, adrenals, thyroid, lungs, heart, great vessels,    oesophagus, stomach, small intestine, spleen, thymus, pancreas,    brain, eye, spinal cord, body wall, pelvis and lower limb.-   Adult tissues examined: Liver, kidney, adrenal, myocardium, aorta,    spleen, lymph node, pancreas, lung, skin, cerebral cortex (rm),    hippocampus(rm), cerebellum(rm), penis, eye, bladder, stomach,    gastric carcinoma, colon, colonic carcinoma, thyroid (chimp),    parathyroid (chimp) ovary (chimp) and chondrosarcoma. Acetominophen    induced liver injury and hepatic cirrhosis    (7) DNA33089-1132 (PRO221)

Specific expression over fetal cerebral white and grey matter, as wellas over neurones in the spinal cord. Probe appears to cross react withrat. Low level of expression over cerebellar neurones in adult rhesusbrain. All other tissues negative.

-   Fetal tissues examined (E12-E16 weeks) include: Placenta, umbilical    cord, liver, kidney, adrenals, thyroid, lungs, heart, great vessels,    oesophagus, stomach, small intestine, spleen, thymus, pancreas,    brain, eye, spinal cord, body wall, pelvis and lower limb.-   Adult tissues examined: Liver, kidney, adrenal, myocardium, aorta,    spleen, lymph node, pancreas, lung, skin, cerebral cortex (rm),    hippocampus(rm), cerebellum(rm), penis, eye, bladder, stomach,    gastric carcinoma, colon, colonic carcinoma and chondrosarcoma.    Acetominophen induced liver injury and hepatic cirrhosis    (8) DNA35918-1174 (PRO258)

Strong expression in the nervous system. In the rhesus monkey brainexpression is observed in cortical, hippocampal and cerebellar neurones.Expression over spinal neurones in the fetal spinal cord, the developingbrain and the inner aspects of the fetal retina. Expression overdeveloping dorsal root and autonomic ganglia as well as enteric nerves.Expression observed over ganglion cells in the adult prostate. In therat, there is strong expression over the developing hind brain andspinal cord. Strong expression over interstitial cells in the placentalvilli. All other tissues were negative.

-   Fetal tissues examined (E12-E16 weeks) include: Placenta, umbilical    cord, liver, kidney, adrenals, thyroid, lungs, heart, great vessels,    oesophagus, stomach, small intestine, spleen, thymus, pancreas,    brain, eye, spinal cord, body wall, pelvis and lower limb.-   Adult tissues examined: Liver, kidney, renal cell carcinoma,    adrenal, aorta, spleen, lymph node, pancreas, lung, myocardium,    skin, cerebral cortex (rm), hippocampus(rm), cerebellum(rm),    bladder, prostate, stomach, gastric carcinoma, colon, colonic    carcinoma, thyroid (chimp), parathyroid (chimp) ovary (chimp) and    chondrosarcoma. Acetominophen induced liver injury and hepatic    cirrhosis.    (9) DNA32286-1191 (PRO214)

Fetal tissue: Low level throughout mesenchyme. Moderate expression inplacental stromal cells in membranous tissues and in thyroid. Low levelexpression in cortical neurones. Adult tissue: all negative.

-   Fetal tissues examined (E12-E16 weeks) include: Placenta, umbilical    cord, liver, kidney, adrenals, thyroid, lungs, heart, great vessels,    oesophagus, stomach, small intestine, spleen, thymus, pancreas,    brain, eye, spinal cord, body wall, pelvis and lower limb.-   Adult tissues examined include: Liver, kidney, adrenal, myocardium,    aorta, spleen, lymph node, pancreas, lung and skin.    (10) DNA33221-1133 (PRO224)

Expression limited to vascular endothelium in fetal spleen, adultspleen, fetal liver, adult thyroid and adult lymph node (chimp).Additional site of expression is the developing spinal ganglia. Allother tissues negative.

-   Human fetal tissues examined (E12-E16 weeks) include: Placenta,    umbilical cord, liver, kidney, adrenals, thyroid, lungs, heart,    great vessels, oesophagus, stomach, small intestine, spleen, thymus,    pancreas, brain, eye, spinal cord, body wall, pelvis and lower limb.-   Adult human tissues examined: Kidney (normal and end-stage),    adrenal, myocardium, aorta, spleen, lymph node, pancreas, lung,    skin, eye (inc. retina), bladder, liver (normal, cirrhotic, acute    failure).-   Non-human primate tissues examined:    -   Chimp Tissues: Salivary gland, stomach, thyroid, parathyroid,        skin, thymus, ovary, lymph node.    -   Rhesus Monkey Tissues: Cerebral cortex, hippocampus, cerebellum,        penis.        (11) DNA35557-1137 (PRO234)

Specific expression over developing motor neurones in ventral aspect ofthe fetal spinal cord (will develop into ventral horns of spinal cord).All other tissues negative. Possible role in growth, differentiationand/or development of spinal motor neurons.

-   Fetal tissues examined (E12-E16 weeks) include: Placenta, umbilical    cord, liver, kidney, adrenals, thyroid, lungs, heart, great vessels,    oesophagus, stomach, small intestine, spleen, thymus, pancreas,    brain, eye, spinal cord, body wall, pelvis and lower limb.-   Adult tissues examined: Liver, kidney, adrenal, myocardium, aorta,    spleen, lymph node, pancreas, lung, skin, cerebral cortex (rm),    hippocampus(rm), cerebellum(rm), penis, eye, bladder, stomach,    gastric carcinoma, colon, colonic carcinoma and chondrosarcoma.    Acetominophen induced liver injury and hepatic cirrhosis    (12) DNA33100-1159 (PRO229)

Striking expression in mononuclear phagocytes (macrophages) of fetal andadult spleen, liver, lymph node and adult thymus (in tingible bodymacrophages). The highest expression is in the spleen. All other tissuesnegative. Localisation and homology are entirely consistent with a roleas a scavenger receptor for cells of the reticuloendothelial system.Expression also observed in placental mononuclear cells.

-   Human fetal tissues examined (E12-E16 weeks) include: Placenta,    umbilical cord, liver, kidney, adrenals, thyroid, lungs, heart,    great vessels, oesophagus, stomach, small intestine, spleen, thymus,    pancreas, brain, eye, spinal cord, body wall, pelvis and lower limb.-   Adult human tissues examined: Kidney (normal and end-stage),    adrenal, myocardium, aorta, spleen, lymph node, gall bladder,    pancreas, lung, skin, eye (inc. retina), prostate, bladder, liver    (normal, cirrhotic, acute failure).-   Non-human primate tissues examined:    -   Chimp Tissues: Salivary gland, stomach, thyroid, parathyroid,        skin, thymus, ovary, lymph node.    -   Rhesus Monkey Tissues: Cerebral cortex, hippocampus, cerebellum,        penis.        (13) DNA34431-1177 (PRO263)

Widepread expression in human fetal tissues and placenta overmononuclear cells, probably macrophages +/−lymphocytes. The cellulardistribution follows a perivascular pattern in many tissues. Strongexpression also seen in epithelial cells of the fetal adrenal cortex.All adult tissues were negative.

-   Fetal tissues examined (E12-E16 weeks) include: Placenta, umbilical    cord, liver, kidney, adrenals, thyroid, lungs, heart, great vessels,    oesophagus, stomach, small intestine, spleen, thymus, pancreas,    brain, eye, spinal cord, body wall, pelvis and lower limb.-   Adult tissues examined: Liver, kidney, adrenal, spleen, lymph node,    pancreas, lung, skin, cerebral cortex (rm), hippocampus(rm),    cerebellum(rm), bladder, stomach, colon and colonic carcinoma.    Acetominophen induced liver injury and hepatic cirrhosis.

A secondary screen evidenced expression over stromal mononuclear cellsprobably histiocytes.

(14) DNA38268-1188 (PRO295)

High expression over ganglion cells in human fetal spinal ganglia andover large neurones in the anterior horns of the developing spinal cord.In the adult there is expression in the chimp adrenal medulla (neural),neurones of the rhesus monkey brain (hippocampus [+++] and cerebralcortex) and neurones in ganglia in the normal adult human prostate (theonly section that contains ganglion cells, expression in this cell typeis presumed NOT to be confined to the prostate). All other tissuesnegative.

-   Human fetal tissues examined (E12-E16 weeks) include: Placenta,    umbilical cord, liver, kidney, adrenals, thyroid, lungs, great    vessels, stomach, small intestine, spleen, thymus, pancreas, brain,    eye, spinal cord, body wall, pelvis, testis and lower limb.-   Adult human tissues examined: Kidney (normal and end-stage),    adrenal, spleen, lymph node, pancreas, lung, eye (inc. retina),    bladder, liver (normal, cirrhotic, acute failure).-   Non-human Primate tissues examined:    -   Chimp Tissues: adrenal    -   Rhesus Monkey Tissues: Cerebral cortex, hippocampus, cerebellum.

Example 103 Isolation of cDNA Clones Encoding Human PRO1868

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA49803. Based up an observed homology between theDNA49803 consensus sequence and an EST sequence contained within theIncyte EST clone no. 2994689, Incyte EST clone no. 2994689 was purchasedand its insert obtained and sequenced. The sequence of that insert isshown in FIG. 123 and is herein designated DNA77624-2515.

The entire nucleotide sequence of DNA77624-2515 is shown in FIG. 123(SEQ ID NO:422). Clone DNA77624-2515 contains a single open readingframe with an apparent translational initiation site at nucleotidepositions 51-53 and ending at the stop codon at nucleotide positions981-983 (FIG. 123). The predicted polypeptide precursor is 310 aminoacids long (FIG. 124). The full-length PRO1868 protein shown in FIG. 124has an estimated molecular weight of about 35,020 daltons and a pI ofabout 7.90. Analysis of the full-length PRO1868 sequence shown in FIG.124 (SEQ ID NO:423) evidences the presence of the following: a signalpeptide from about amino acid 1 to about amino acid 30, a transmembranedomain from about amino acid 243 to about amino acid 263, potentialN-glycosylation sites from about amino acid 104 to about amino acid 107and from about amino acid 192 to about amino acid 195, a cAMP- andcGMP-dependent protein kinase phosphorylation site from about amino acid107 to about amino acid 110, casein kinase II phosphorylation sites fromabout amino acid 106 to about amino acid 109 and from about amino acid296 to about amino acid 299, a tyrosine kinase phosphorylation site fromabout amino acid 69 to about amino acid 77 and potential N-myristolationsites from about amino acid 26 to about amino acid 31, from about aminoacid 215 to about amino acid 220, from about amino acid 226 to aboutamino acid 231, from about amino acid 243 to about amino acid 248, fromabout amino acid 244 to about amino acid 249 and from about amino acid262 to about amino acid 267. Clone DNA77624-2515 has been deposited withATCC on Dec. 22, 1998 and is assigned ATCC deposit no. 203553.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 124 (SEQ ID NO:423), evidenced significant homologybetween the PRO1868 amino acid sequence and the following Dayhoffsequences: HGS_RC75, P_W61379, A33_HUMAN, P_W14146, P_W14158,AMAL_DROME, P_R77437, I38346, NCM2_HUMAN and PTPD_HUMAN.

Example 104 Identification of Receptor/Ligand Interactions

In this assay, various PRO polypeptides are tested for ability to bindto a panel of potential receptor molecules for the purpose ofidentifying receptor/ligand interactions. The identification of a ligandfor a known receptor, a receptor for a known ligand or a novelreceptor/ligand pair is useful for a variety of indications including,for example, targeting bioactive molecules (linked to the ligand orreceptor) to a cell known to express the receptor or ligand, use of thereceptor or ligand as a reagent to detect the presence of the ligand orreceptor in a composition suspected of containing the same, wherein thecomposition may comprise cells suspected of expressing the ligand orreceptor, modulating the growth of or another biological orimmunological activity of a cell known to express or respond to thereceptor or ligand, modulating the immune response of cells or towardcells that express the receptor or ligand, allowing the preparaion ofagonists, antagonists and/or antibodies directed against the receptor orligand which will modulate the growth of or a biological orimmunological activity of a cell expressing the receptor or ligand, andvarious other indications which will be readily apparent to theordinarily skilled artisan.

The assay is performed as follows. A PRO polypeptide of the presentinvention suspected of being a ligand for a receptor is expressed as afusion protein containing the Fc domain of human IgG (an immunoadhesin).Receptor-ligand binding is detected by allowing interaction of theimmunoadhesin polypeptide with cells (e.g. Cos cells) expressingcandidate PRO polypeptide receptors and visualization of boundimmunoadhesin with fluorescent reagents directed toward the Fc fusiondomain and examination by microscope. Cells expressing candidatereceptors are produced by transient transfection, in parallel, ofdefined subsets of a library of cDNA expression vectors encoding PROpolypeptides that may function as receptor molecules. Cells are thenincubated for 1 hour in the presence of the PRO polypeptideimmunoadhesin being tested for possible receptor binding. The cells arethen washed and fixed with paraformaldehyde. The cells are thenincubated with fluorescent conjugated antibody directed against the Fcportion of the PRO polypeptide immunoadhesin (e.g. FITC conjugated goatanti-human-Fc antibody). The cells are then washed again and examined bymicroscope. A positive interaction is judged by the presence offluorescent labeling of cells transfected with cDNA encoding aparticular PRO polypeptide receptor or pool of receptors and an absenceof similar fluorescent labeling of similarly prepared cells that havebeen transfected with other cDNA or pools of cDNA. If a defined pool ofcDNA expression vectors is judged to be positive for interaction with aPRO polypeptide immunoadhesin, the individual cDNA species that comprisethe pool are tested individually (the pool is “broken down”) todetermine the specific cDNA that encodes a receptor able to interactwith the PRO polypeptide immunoadhesin.

In another embodiment of this assay, an epitope-tagged potential ligandPRO polypeptide (e.g. 8 histidine “His” tag) is allowed to interact witha panel of potential receptor PRO polypeptide molecules that have beenexpressed as fusions with the Fc domain of human IgG (immunoadhesins).Following a 1 hour co-incubation with the epitope tagged PROpolypeptide, the candidate receptors are each immunoprecipitated withprotein A beads and the beads are washed. Potential ligand interactionis determined by western blot analysis of the immunoprecipitatedcomplexes with antibody directed towards the epitope tag. An interactionis judged to occur if a band of the anticipated molecular weight of theepitope tagged protein is observed in the western blot analysis with acandidate receptor, but is not observed to occur with the other membersof the panel of potential receptors.

Using these assays, the following receptor/ligand interactions have beenherein identified: PRO245 binds to PRO1868.

Deposit of Material

The following materials have been deposited with the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md., USA (ATCC):

Material ATCC Dep. No. Deposit Date DNA32292-1131 ATCC 209258 Sep. 16,1997 DNA33094-1131 ATCC 209256 Sep. 16, 1997 DNA33223-1136 ATCC 209264Sep. 16, 1997 DNA34435-1140 ATCC 209250 Sep. 16, 1997 DNA27864-1155 ATCC209375 Oct. 16, 1997 DNA36350-1158 ATCC 209378 Oct. 16, 1997DNA32290-1164 ATCC 209384 Oct. 16, 1997 DNA35639-1172 ATCC 209396 Oct.17, 1997 DNA33092-1202 ATCC 209420 Oct. 28, 1997 DNA49435-1219 ATCC209480 Nov. 21, 1997 DNA35638-1141 ATCC 209265 Sep. 16, 1997DNA32298-1132 ATCC 209257 Sep. 16, 1997 DNA33089-1132 ATCC 209262 Sep.16, 1997 DNA33786-1132 ATCC 209253 Sep. 16, 1997 DNA35918-1174 ATCC209402 Oct. 17, 1997 DNA37150-1178 ATCC 209401 Oct. 17, 1997DNA38260-1180 ATCC 209397 Oct. 17, 1997 DNA39969-1185 ATCC 209400 Oct.17, 1997 DNA32286-1191 ATCC 209385 Oct. 16, 1997 DNA33461-1199 ATCC209367 Oct. 15, 1997 DNA40628-1216 ATCC 209432 Nov. 7, 1997DNA33221-1133 ATCC 209263 Sep. 16, 1997 DNA33107-1135 ATCC 209251 Sep.16, 1997 DNA35557-1137 ATCC 209255 Sep. 16, 1997 DNA34434-1139 ATCC209252 Sep. 16, 1997 DNA33100-1159 ATCC 209373 Oct. 16, 1997DNA35600-1162 ATCC 209370 Oct. 16, 1997 DNA34436-1238 ATCC 209523 Dec.10, 1997 DNA33206-1165 ATCC 209372 Oct. 16, 1997 DNA35558-1167 ATCC209374 Oct. 16, 1997 DNA35599-1168 ATCC 209373 Oct. 16, 1997DNA36992-1168 ATCC 209382 Oct. 16, 1997 DNA34407-1169 ATCC 209383 Oct.16, 1997 DNA35841-1173 ATCC 209403 Oct. 17, 1997 DNA33470-1175 ATCC209398 Oct. 17, 1997 DNA34431-1177 ATCC 209399 Oct. 17, 1997DNA39510-1181 ATCC 209392 Oct. 17, 1997 DNA39423-1182 ATCC 209387 Oct.17, 1997 DNA40620-1183 ATCC 209388 Oct. 17, 1997 DNA40604-1187 ATCC209394 Oct. 17, 1997 DNA38268-1188 ATCC 209421 Oct. 28, 1997DNA37151-1193 ATCC 209393 Oct. 17, 1997 DNA35673-1201 ATCC 209418 Oct.28, 1997 DNA40370-1217 ATCC 209485 Nov. 21, 1997 DNA42551-1217 ATCC209483 Nov. 21, 1997 DNA39520-1217 ATCC 209482 Nov. 21, 1997DNA41225-1217 ATCC 209491 Nov. 21, 1997 DNA43318-1217 ATCC 209481 Nov.21, 1997 DNA40587-1231 ATCC 209438 Nov. 7, 1997 DNA41338-1234 ATCC209927 Jun. 2, 1998 DNA40981-1234 ATCC 209439 Nov. 7, 1997 DNA37140-1234ATCC 209489 Nov. 21, 1997 DNA40982-1235 ATCC 209433 Nov. 7, 1997DNA41379-1236 ATCC 209488 Nov. 21, 1997 DNA44167-1243 ATCC 209434 Nov.7, 1997 DNA39427-1179 ATCC 209395 Oct. 17, 1997 DNA40603-1232 ATCC209486 Nov. 21, 1997 DNA43466-1225 ATCC 209490 Nov. 21, 1997DNA43046-1225 ATCC 209484 Nov. 21, 1997 DNA35668-1171 ATCC 209371 Oct.16, 1997 DNA77624-2515 ATCC 203553 Dec. 22, 1998

These deposit were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposits will be made availableby ATCC under the terms of the Budapest Treaty, and subject to anagreement between Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the culture of the depositto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trademarks to be entitledthereto according to 35 USC §122 and the Commissioner's rules pursuantthereto (including 37 CFR §1.14 with particular reference to 886 OG638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct deposited,since the deposited embodiment is intended as a single illustration ofcertain aspects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1. An isolated nucleic acid comprising: (a) a nucleic acid sequenceencoding the polypeptide shown in FIG. 15 (SEQ ID NO: 34); (b) a nucleicacid sequence encoding the polypeptide shown in FIG. 15 (SEQ ID NO: 34),lacking its associated signal peptide; (c) the full-length codingsequence of the nucleic acid sequence shown in FIG. 14 (SEQ ID NO: 33);or (d) the full-length coding sequence of the cDNA deposited under ATCCaccession number
 209384. 2. The isolated nucleic acid of claim 1comprising a nucleic acid sequence encoding the polypeptide shown inFIG. 15 (SEQ ID NO: 34).
 3. The isolated nucleic acid of claim 1comprising a nucleic acid sequence encoding the polypeptide shown inFIG. 15 (SEQ ID NO: 34), lacking its associated signal peptide.
 4. Theisolated nucleic acid of claim 1 comprising the nucleic acid sequenceshown in FIG. 14 (SEQ ID NO: 33).
 5. The isolated nucleic acid of claim1 comprising the sequence from nucleotide 204 to nucleotide 2949 of thenucleic acid sequence shown in FIG. 14 (SEQ ID NO: 33).
 6. The isolatednucleic acid of claim 1 comprising the full-length coding sequence ofthe cDNA deposited under ATCC accession number
 209384. 7. A vectorcomprising the nucleic acid of claim
 1. 8. The vector of claim 7,wherein said nucleic acid is operably linked to control sequencesrecognized by a host cell transformed with the vector.
 9. An isolatedhost cell comprising the vector of claim
 7. 10. The host cell of claim9, wherein said cell is a CHO cell, an E. coli or a yeast cell.